Modular universal plasmid design strategy for the assembly and editing of multiple dna constructs for multiple hosts

ABSTRACT

Methods, compositions, and kits for high throughput DNA assembly reactions in vitro. Modular CRISPR DNA constructs comprising modular insert DNA parts flanked by cloning tag segments comprising pre-validated CRISPR protospacer/protospacer adjacent motif sequence combinations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application a Divisional of U.S. application Ser. No. 16/535,245,filed on Aug. 8, 2019, which is a Continuation U.S. Utility Applicationunder 35 U.S.C. § 111, which claims the benefit of International PCTApplication No. PCT/US2018/017573, filed on Feb. 9, 2018, which itselfclaims the benefit of priority to U.S. Provisional Application No.62/457,493, filed on Feb. 10, 2017, each of which is hereby incorporatedby reference in its entirety for all purposes.

GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under Agreement No.HR0011-15-9-0014, awarded by DARPA. The Government has certain rights inthe invention.

FIELD

The present disclosure relates to systems, methods, and compositionsused for guided genetic sequence editing in vitro. The disclosuredescribes, inter alia, methods of using guided sequence editingcomplexes for improved DNA cloning, assembly of oligonucleotides, andfor the improvement of microorganisms.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ZYMR_010_01WO_SeqList_ST25.txt, date recorded: Jan. 30, 2018; file size:817,847 bytes).

BACKGROUND

A major area of interest in biology is the in vitro and in vivo targetedmodification of genetic sequences. Indeed, one of the most significantbottlenecks to academic and commercial genetic research has been thespeed with which novel genetic constructs could be generated or latermodified prior to testing.

The currently available cloning techniques relying on restriction siterecognition or DNA hybridization and amplification have proven to beslow, unreliable, and intractable to later modifications. The discoveryof Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)gene editing systems have provided researchers with additional avenuesfor genetic modification. Even these new approaches, however, remainimpractical for high throughput modular cloning applications.

CRISPR editing locations for example, are often limited by the locationof protospacer adjacent motifs (PAMs). De novo CRISPR guide RNA designand gene targeting can be both time consuming and expensive, and is alsosusceptible to low efficiencies, and potential for off-target mutations.

Thus, there is a need for improved compositions and methods for targetedalteration of genetic sequences.

SUMMARY OF THE DISCLOSURE

In some embodiments, the present disclosure teaches methods,compositions, and kits for high-throughput DNA assembly reactions invivo and in vitro utilizing modular CRISPR DNA constructs.

Thus, in some embodiments, the present disclosure teaches CRISPR DNAconstructs comprising modular insert DNA parts flanked by cloning tagsegments comprising pre-validated CRISPR protospacer/protospaceradjacent motif (PAM) sequence combinations. In some embodiments thepresent disclosure teaches digesting DNA with CRISPR endonucleases. Insome embodiments, the present disclosure teaches digesting DNA with TypeII-Class 2 CRISPR endonucleases (e.g. Cas9). In some embodiments, thepresent disclosure teaches digesting DNA with Type V-Class 2 CRISPRendonucleases. In some embodiments, the present disclosure teachesdigesting DNA with Cpf1 endonucleases.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct comprising a CRISPR multi-clonal site, saidmulti-clonal site comprising: a) at least two distinct cloning tags(cTAG), wherein each cTAG comprises: i) one or more validated CRISPRlanding sites, each comprising a protospacer sequence operably linked toa protospacer adjacent motif (PAM); wherein at least one of saidvalidated CRISPR landing sites is unique within the modular CRISPR DNAconstruct; and b) one or more DNA insert sequences; i) wherein each ofsaid cTAGs are distributed in flanking positions around each of the oneor more DNA insert sequences; and ii) wherein at least one of said DNAinsert sequences comprises a selection marker.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct, wherein said modular CRISPR DNA constructis circular.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct, wherein said modular CRISPR DNA constructis linear.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct, wherein said modular CRISPR DNA constructis integrated into the genome of an organism.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct, wherein at least one of said distinctcTAGs comprises at least two validated CRISPR landing sites.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct, wherein at least one of the CRISPR landingsites is for a Cas9 endonuclease.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct, wherein at least one of the CRISPR landingsites is for a Cpf1 endonuclease.

In some embodiments, the present disclosure teaches a recombinantmodular CRISPR DNA construct, wherein at least one of said distinctcTAGs comprises a rare (>8 bases long) restriction endonuclease site.

In some aspects, the disclosure refers to a recombinant modular CRISPRDNA construct as a “MegaModular” construct.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, the method comprising: a)forming a mixture comprising: i) a plurality of DNA insert parts,wherein each DNA insert part is flanked by two cloning tags (cTAGs),each cTAG comprising: 1) one or more validated CRISPR landing sites,each comprising a protospacer sequence operably linked to a protospaceradjacent motif (PAM); ii) one or more CRISPR complexes targeting atleast one of said cTAGs present in at least two of the plurality of DNAinsert parts, each CRISPR complex comprising; 1) a CRISPR endonuclease,and 2) a guide RNA or guide RNAs capable of recruiting said CRISPRendonuclease to one of said targeted cTAGs; wherein the mixture isincubated under conditions which allow for digestion of the targetedcTAG(s) in at least two of the plurality of DNA insert parts to generateoverhanging ends, and b) incubating the digestion products generated in(a) in conditions which allow for hybridization of compatibleoverhanging ends and covalent joining of the hybridized ends; whereinthe resulting recombinant nucleic acid molecule comprises the completecTAG sequences of the original insert parts that are ligated in themethod.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, wherein the CRISPRendonuclease is Cpf1.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, wherein the CRISPRendonuclease is Cas9.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, wherein the methodcomprises the step of: i) separating the digested cTAG sequences fromthe CRISPR complexes prior to ligation, or ii) inactivating the CRISPRcomplexes prior to ligation.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, wherein the separationstep comprises a DNA purification step.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, wherein the inactivationstep comprises heat or chemical inactivation of said CRISPR complexes.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, wherein the two cTAGs foreach of the plurality of DNA insert parts form a cTAG pair, and whereinsaid cTAG pair is unique from all other cTAG pairs of the DNA insertparts that are ligated in the method.

In some embodiments, the present disclosure teaches a method forpreparing a recombinant nucleic acid molecule, wherein at least one ofthe cTAGs in each cTAG pair is the same as at least one other cTAG in adifferent cTAG pair.

In some embodiments, the present disclosure teaches a method for DNAsequence editing, said method comprising: a) introducing into areaction: i) the modular CRISPR DNA construct of the present disclosure:ii) a replacement DNA insert part, wherein said replacement DNA insertpart is flanked by a first and second insert cTAG; 1) wherein the firstinsert cTAG comprises the validated CRISPR landing site(s) of one of thedistinct cTAGs of the modular CRISPR DNA construct, and the secondinsert cTAG comprises the validated CRISPR landing site(s) of anotherdistinct cTAG of the modular CRISPR DNA construct; and iii) a first andsecond CRISPR complex targeting the first and second insert cTAGs,respectively, each CRISPR complex comprising: 1) a CRISPR endonuclease,and 2) a guide RNA capable of recruiting said CRISPR endonuclease to oneof said targeted insert cTAGs; wherein the first and second CRISPRcomplexes cleave the first and second insert cTAGs and theircorresponding distinct cTAGs to generate overhanging ends, and b)incubating the replacement DNA insert part and modular CRISPR DNAconstruct with digested cTAGs generated: (a) under conditions whichallow for hybridization of compatible overhanging ends and covalentjoining of the hybridized ends; wherein the resulting edited modularCRISPR DNA construct comprises the complete cTAG sequences of theoriginal insert part that is ligated by the method.

In some embodiments, the present disclosure teaches a method for DNAsequence editing, wherein the reaction of step (b) comprises afunctional ligase.

In some embodiments, the present disclosure teaches a method for DNAsequence editing, said method comprising: a) introducing into areaction: i) the modular CRISPR DNA construct of the present disclosure;ii) at least two CRISPR complexes targeting two distinct cTAGs in themodular CRISPR DNA construct, each CRISPR complex comprising; 1) aCRISPR endonuclease, and 2) a guide RNA capable of recruiting saidCRISPR endonuclease to one of said targeted distinct cTAGs; wherein thefirst and second CRISPR complexes cleave the two distinct cTAGs in themodular CRISPR DNA construct, wherein the resulting distinct cTAGscomprise overhang ends, and b) introducing into a second reaction: i)the modular CRISPR DNA construct with digested cTAGs generated in (a);and ii) a replacement DNA insert part, wherein said replacement DNAinsert part is flanked by a first and second insert cTAG; 1) wherein thefirst insert cTAG comprises the polynucleotide sequence of one of theundigested distinct cTAGs that is cleaved in (a), and the second insertcTAG comprises the polynucleotide sequence of another undigesteddistinct cTAG that is cleaved in (a); and 2) wherein the first andsecond insert cTAGs comprise overhang ends that are compatible with theoverhang ends of the distinct cTAGs from (a); under conditions whichallow for hybridization of compatible the overhanging ends and covalentjoining of the hybridized ends; wherein the resulting edited modularCRISPR DNA construct comprises the complete sequences of the originalundigested distinct cTAGs that were targeted in (a).

In some embodiments, the present disclosure teaches a method for DNAsequence editing, wherein the reaction of step (b) comprises afunctional ligase.

In some embodiments, the present disclosure teaches a method for DNAsequence editing, wherein the CRISPR endonuclease is Cpf1.

In some embodiments, the present disclosure teaches a method for DNAsequence editing, wherein step (a) further comprises digesting the twocleaved distinct cTAGs with a single stranded exonuclease, therebyproducing the distinct cTAGs with overhang ends. In some aspects, onemay add a ligase and polymerase to repair the junctions with apolymerase and ligase after the exonuclease step. In some aspects, thisreaction can also be done with Cas9 digested, blunt-end cuts.

In some embodiments, the present disclosure teaches a method for DNAsequence editing, wherein the CRISPR endonuclease is Cas9.

In some embodiments, the disclosure provides for a host cell genomecomprising a recombinant modular CRISPR DNA construct comprising aCRISPR multi-clonal site, said multi-clonal site comprising: a) at leasttwo distinct cloning tag (cTAG), wherein each cTAG comprises: i) one ormore validated CRISPR landing sites, each comprising a protospacersequence operably linked to a protospacer adjacent motif (PAM); whereinat least one of said validated CRISPR landing sites is unique within themodular CRISPR DNA construct; and b) one or more DNA insert part(s); i)wherein each of said distinct cTAGs are distributed in flankingpositions around each of the one or more DNA insert part(s).

In some embodiments, the disclosure provides for a method for preparinga recombinant nucleic acid molecule, the method comprising: a)incubating a mixture comprising: i) a plurality of DNA insert partsflanked by two cloning tags (cTAGs), each cTAG comprising: 1) one ormore validated CRISPR landing sites, each comprising a protospacersequence operably linked to a protospacer adjacent motif (PAM); and 2) arare (≥8 base) restriction enzyme recognition site; wherein at least oneof the cTAGs of at least two insert parts comprise the same restrictionenzyme site; ii) one or more restriction enzymes targeting the rarerestriction enzyme sites in at least two of the plurality of DNA insertparts; under conditions which allow for digestion of the targeted cTAGby the one or more restriction enzymes in at least two of the pluralityof DNA insert parts to generate insert parts with digested DNA ends; andb) incubating the DNA insert part(s) with digested DNA ends generated instep (a) under conditions which allow for the covalent joining of thedigested DNA ends; wherein the resulting recombinant nucleic acidmolecule comprises the complete cTAG sequences of the original insertparts that are covalently joined in the method.

In some embodiments, the disclosure provides for a method for DNAsequence editing, said method comprising: a) providing: i) the modularCRISPR DNA construct of claim 1, wherein at least two of the distinctcTAGs comprise a rare (≥8 base) restriction enzyme recognition site; ii)a replacement DNA insert part, wherein said replacement DNA insert partis flanked by a first and second insert cTAG; 1) wherein the firstinsert cTAG comprises the rare restriction enzyme recognition site ofone of the distinct cTAGs of the modular CRISPR DNA construct, and thesecond insert cTAG comprises the rare restriction enzyme recognitionsite of another distinct cTAG of the modular CRISPR DNA construct; andiii) one or more restriction enzymes targeting the rare restrictionenzyme sites in the first and second insert cTAGs; wherein parts (i) and(ii) are each incubated with part (iii) in a single or separatereactions; wherein the one or more restriction enzymes cleave the rarerestriction enzyme recognition sites of first and second insert cTAGsand their corresponding distinct cTAGs to generate digested DNA ends,and b) incubating the replacement DNA insert part and modular CRISPR DNAconstruct with digested DNA ends generated in step (a) under conditionswhich allow for the covalent joining of the digested DNA ends; whereinthe resulting edited modular CRISPR DNA construct comprises the completecTAG sequences of the original insert part that is covalently joined bythe method.

In some embodiments, the disclosure provides for a method for preparinga recombinant nucleic acid molecule, the method comprising: a)incubating a mixture comprising: i) a plurality of DNA insert parts,wherein each DNA insert part is flanked by two cloning tags (cTAGs),each cTAG comprising: 1) one or more validated CRISPR landing sites,each comprising a protospacer sequence operably linked to a protospaceradjacent motif (PAM); wherein at least two of the DNA insert parts sharethe same cTAG; ii) a single stranded DNA (ssDNA) exonuclease; underconditions which allow for digestion of the shared cTAG in the least twoDNA insert parts, thereby generating compatible overhang DNA ends in theat least two DNA insert parts, and b) incubating the DNA insert partswith digested cTAGs generated in (a) under conditions which allow forthe hybridization and covalent joining of the compatible overhang DNAends of the least two DNA insert parts; wherein the resultingrecombinant nucleic acid molecule comprises the complete cTAG sequencesof the shared cTAG before digestion. This reaction can also be conductedwith a polymerase and or ligase that are used to fix junctions. Further,this can be carried out with a predigested vector.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C illustrates a comparison of the CRISPR/Cas9 and CRISPR/Cpf1systems of the present disclosure. FIG. 1A—Cas9 endonucleases arerecruited to target dsDNA by tracrRNA and crRNA complexes. FIG. 1B—Cas9endonucleases may also be recruited to target dsDNA by artificiallyfused tracrRNA and crRNA sequences known as single-guide RNAs (sgRNAs).Cas9 endonuc lease produces blunt ends. FIG. 1C—Cpf1 endonucleases onlyrequire crRNA guide poly-ribonucleotides. Cpf1 endonuclease cleavageproduces double stranded breaks with 5′ overhangs.

FIGS. 2A-2C illustrates an embodiment of the present cloning methodsutilizing modular CRISPR constructs of the present disclosure. FIG.2A—diagrams a modular CRISPR plasmid that can be easily altered withCas9 or Cpf1 nucleases, according to the present disclosure. Asaforementioned, the modular CRISPR constructs of the disclosure can betermed “MegaModular” constructs. Interchangeable parts represented bynumbers are flanked by invariant cTAG sequences represented by letters.Parts may come pre-assembled, or may be assembled in vitro based on cTAGsequence identity. Example insert parts are shown on the right of FIG.2A. FIG. 2B—Several strategies such as Cas9, Cpf1, or restrictionendonuclease cleavage at cTAGs may be used to replace individual partswithout having to reassemble the entire plasmid. cTAG sequences maycomprise one or more cloning sites, including, but not limited to Cas9,Cpf1, restriction, and/or recombination sites. FIG. 2C—Once integratedinto the genome of an organism, cTAGs may continue to serve aspre-validated Cas9 or Cpf1 landing sites, enabling replacement,insertion, or removal of genomically integrated DNA with prevalidatedand orthogonal gRNA sequences.

FIGS. 3A-3D illustrates an embodiment of the present cloning methodsutilizing modular CRISPR constructs of the present disclosure. FIG.3A—diagrams a modular CRISPR plasmid that can be easily altered withCas9 or Cpf1 nucleases, according to present disclosure. Interchangeableparts represented by numbers are flanked by invariant cTAG sequencesrepresented by letters. Parts may come pre-assembled, or may beassembled in vivo or in vitro based on cTAG sequence identity. Exampleinsert parts are shown on the right of FIG. 3A. FIG. 3B—Severalstrategies such as Cas9, Cpf1, or restriction endonuclease cleavage atcTAGs may be used to replace individual parts without having toreassemble the entire plasmid. cTAG sequences may comprise one or morecloning sites, including, but not limited to Cas9, Cpf1, restriction,and/or recombination sites. FIG. 3C—Illustrates methods of the presentdisclosure for removing insert parts, or for adding stuffer sequencesfrom existing modular plasmids. FIG. 3D—Insert parts of the modularplasmids of the present disclosure may serve as sequences for genomicintegration of a portion or the whole of the modular CRISPR vectors intothe genome of a host cell.

FIG. 4 illustrates the one-pot in vitro modular CRISPR cloning ofExample 1. Specifically, the generation of plasmid 13001009086 bytransfer of an insert from one plasmid to another in a one-pot reactionis shown. The details of this reaction are set forth in Example 1.

FIG. 5 illustrates an embodiment of the in vitro modular CRISPR cloningmethods of Example 2. Each panel provides an illustration of theexperimental design described in Example 2. A chloramphenicol resistancegene was cloned into a kanamycin resistant backbone plasmid to create adual resistance plasmid. Dual resistance plasmids were then transformedinto bacteria, which was subsequently cultured in media augmented withkanamycin and chloramphenicol antibiotics. Resistant colonies indicatedsuccessful Cpf1 cloning assemblies.

FIG. 6 illustrates the results of the in vitro modular CRISPR cloningmethods of Example 2. The y-axis represents the number of recoveredcolonies growing in media augmented with kanamycin and chloramphenicol.Resistant colonies indicate successful Cpf1 cloning assemblies. Theresults showed a ligase-dependent assembly of dual resistance plasmids.

FIG. 7 Depicts the vector map for pJDI427. CRISPR landing sites used inthe Cpf1assembly are labeled as cTAG M and cTAG N. Relevant sequenceinformation can be found in SEQ ID NO: 102.

FIG. 8 Depicts the vector map for pJDI429. CRISPR landing sites used inthe Cpf1assembly are labeled as cTAG N and cTAG 0. Relevant sequenceinformation can be found in SEQ ID NO: 103.

FIG. 9 Depicts the vector map for pJDI430. CRISPR landing sites used inthe Cpf1assembly are labeled as cTAG P and cTAG N. Relevant sequenceinformation can be found in SEQ ID NO: 104.

FIG. 10 Depicts the vector map for pJDI431. CRISPR landing sites used inthe Cpf1assembly are labeled as cTAG P and cTAG 0. Relevant sequenceinformation can be found in SEQ ID NO: 105.

FIG. 11 Depicts the vector map for pJDI432. CRISPR landing sites used inthe Cpf1assembly are labeled as cTAG M and cTAG N. Relevant sequenceinformation can be found in SEQ ID NO: 106.

FIG. 12 Depicts the vector map for pJDI434. CRISPR landing sites used inthe Cpf1C assembly are labeled as cTAG N and cTAG 0. Relevant sequenceinformation can be found in SEQ ID NO: 107.

FIG. 13 Depicts the vector map for pJDI435. CRISPR landing sites used inthe Cpf1assembly are labeled as cTAG P and cTAG N. Relevant sequenceinformation can be found in SEQ ID NO: 108.

FIG. 14 Depicts the vector map for pJDI436. CRISPR landing sites used inthe Cpf1assembly are labeled as cTAG P and cTAG 0. Relevant sequenceinformation can be found in SEQ ID NO: 109.

FIG. 15 illustrates an example gene editing of a modular CRISPRconstruct, according to the methods of the present disclosure.Specifically, FIG. 15 illustrates a plasmid assembly by restrictionenzyme digestion and ligation using the megamodular design of Example 3.FIG. 15 shows that a modular CRISPR plasmid backbone p1300283391 and acompatible GFP-containing insert DNA part are each digested with ApaIand PvuI restriction enzymes to create compatible cloning tag ends. Thedigested backbone and insert are ligated in vitro to create a newmodular CRISPR construct

DETAILED DESCRIPTION Definitions

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

The term “a” or “an” refers to one or more of that entity, i.e., canrefer to a plural referent. As such, the terms “a” or “an”, “one ormore” and “at least one” are used interchangeably herein. In addition,reference to “an element” by the indefinite article “a” or “an” does notexclude the possibility that more than one of the elements is present,unless the context clearly requires that there is one and only one ofthe elements.

The term “prokaryotes” is art recognized and refers to cells whichcontain no nucleus. The prokaryotes are generally classified in one oftwo domains, the Bacteria and the Archaea. The definitive differencebetween organisms of the Archaea and Bacteria domains is based onfundamental differences in the nucleotide base sequence in the 16Sribosomal RNA.

A “eukaryote” is any organism whose cells contain a nucleus and otherorganelles enclosed within membranes. Eukaryotes belong to the taxonEukarya or Eukaryota. The defining feature that sets eukaryotic cellsapart from prokaryotic cells (the aforementioned Bacteria and Archaea)is that they have membrane-bound organelles, especially the nucleus,which contains the genetic material, and is enclosed by the nuclearenvelope.

The term “Archaea” refers to a categorization of organisms of thedivision Mendosicutes, typically found in unusual environments anddistinguished from the rest of the prokaryotes by several criteria,including the number of ribosomal proteins and the lack of muramic acidin cell walls. On the basis of ssrRNA analysis, the Archaea consist oftwo phylogenetically-distinct groups: Crenarchaeota and Euryarchaeota.On the basis of their physiology, the Archaea can be organized intothree types: methanogens (prokaryotes that produce methane); extremehalophiles (prokaryotes that live at very high concentrations of salt(NaCl)); and extreme (hyper) thermophilus (prokaryotes that live at veryhigh temperatures). Besides the unifying archaeal features thatdistinguish them from Bacteria (i.e., no murein in cell wall,ester-linked membrane lipids, etc.), these prokaryotes exhibit uniquestructural or biochemical attributes which adapt them to theirparticular habitats. The Crenarchaeota consists mainly ofhyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeotacontains the methanogens and extreme halophiles.

“Bacteria” or “eubacteria” refers to a domain of prokaryotic organisms.Bacteria include at least 11 distinct groups as follows: (1)Gram-positive (gram+) bacteria, of which there are two majorsubdivisions: (1) high G+C group (Actinomycetes, Mycobacteria,Micrococcus, others) (2) low G+C group (Bacillus, Clostridia,Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2)Proteobacteria, e.g., Purple photosynthetic +non-photosyntheticGram-negative bacteria (includes most “common” Gram-negative bacteria);(3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes andrelated species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7)Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria(also anaerobic phototrophs); (10) Radioresistant micrococci andrelatives; (11) Thermotoga and Thermosipho thermophiles.

The terms “genetically modified host cell,” “recombinant host cell,” and“recombinant strain” are used interchangeably herein and refer to hostcells that have been genetically modified by the cloning andtransformation methods of the present disclosure. Thus, the termsinclude a host cell (e.g., bacteria, yeast cell, fungal cell, CHO, humancell, etc.) that has been genetically altered, modified, or engineered,such that it exhibits an altered, modified, or different genotype and/orphenotype (e.g., when the genetic modification affects coding nucleicacid sequences of the microorganism), as compared to thenaturally-occurring microorganism from which it was derived. It isunderstood that the terms refer not only to the particular recombinantmicroorganism in question, but also to the progeny or potential progenyof such a microorganism.

The term “genetically engineered” may refer to any manipulation of ahost cell's genome (e.g. by insertion or deletion of nucleic acids).

As used herein, “selectable marker” is a nucleic acid segment thatallows one to select for a molecule (e.g., a replicon) or a cell thatcontains it, often under particular conditions. These markers can encodean activity, such as, but not limited to, production of RNA, peptide, orprotein, or can provide a binding site for RNA, peptides, proteins,inorganic and organic compounds or compositions and the like. Examplesof selectable markers include but are not limited to: (1) nucleic acidsegments that encode products which provide resistance against otherwisetoxic compounds (e.g., antibiotics); (2) nucleic acid segments thatencode products which are otherwise lacking in the recipient cell (e.g.,tRNA genes, auxotrophic markers); (3) nucleic acid segments that encodeproducts which suppress the activity of a gene product; (4) nucleic acidsegments that encode products which can be readily identified (e.g.,phenotypic markers such as β-galactosidase, green fluorescent protein(GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP),and cell surface proteins); (5) nucleic acid segments that encodeproducts that bind other products which are otherwise detrimental tocell survival and/or function; (6) nucleic acid segments that encodenucleic acids that otherwise inhibit the activity of any of the nucleicacid segments resulting in a visible or selectable phenotype (e.g.,antisense oligonucleotides); (7) nucleic acid segments that encodeproducts that bind other products that modify a substrate (e.g.restriction endonucleases); (8) nucleic acid segments that can be usedto isolate or identify a desired molecule (e.g. specific protein bindingsites); (9) nucleic acid segments that encode a specific nucleotidesequence which can be otherwise non-functional (e.g., for PCRamplification of subpopulations of molecules); and (10) nucleic acidsegments, which when absent, directly or indirectly confer resistance orsensitivity to particular compounds.

As used herein, “counterselectable marker” or a “counterselectionmarker” is a nucleic acid segment that eliminates or inhibits growth ofa host organism upon selection. In some embodiments, thecounterselectable markers of the present disclosure render the cellssensitive to one or more chemicals/growth conditions/geneticbackgrounds. In some embodiments, the counterselectable markers of thepresent disclosure are toxic genes. In some embodiments, thecounterselectable markers are expressed by inducible promoters.

As used herein, the term “nucleic acid” refers to a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides, or analogs thereof. This term refers to theprimary structure of the molecule, and thus includes double- andsingle-stranded DNA, as well as double- and single-stranded RNA. It alsoincludes modified nucleic acids such as methylated and/or capped nucleicacids, nucleic acids containing modified bases, backbone modifications,and the like. The terms “nucleic acid” and “nucleotide sequence” areused interchangeably.

As used herein, the term “gene” refers to any segment of DNA associatedwith a biological function. Thus, genes include, but are not limited to,coding sequences and/or the regulatory sequences required for theirexpression. Genes can also include non-expressed DNA segments that, forexample, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

As used herein, the term “homologous” or “homolog” or “ortholog” isknown in the art and refers to related sequences that share a commonancestor or family member and are determined based on the degree ofsequence identity. The terms “homology,” “homologous,” “substantiallysimilar” and “corresponding substantially” are used interchangeablyherein. They refer to nucleic acid fragments wherein changes in one ormore nucleotide bases do not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant disclosure such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the disclosure encompasses more than the specificexemplary sequences. These terms describe the relationship between agene found in one species, subspecies, variety, cultivar or strain andthe corresponding or equivalent gene in another species, subspecies,variety, cultivar or strain. For purposes of this disclosure homologoussequences are compared. “Homologous sequences” or “homologs” or“orthologs” are thought, believed, or known to be functionally related.A functional relationship may be indicated in any one of a number ofways, including, but not limited to: (a) degree of sequence identityand/or (b) the same or similar biological function. Preferably, both (a)and (b) are indicated. Homology can be determined using softwareprograms readily available in the art, such as those discussed inCurrent Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987) Supplement 30, section 7.718, Table 7.71. Some alignment programsare MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus(Scientific and Educational Software, Pennsylvania) and AlignX (VectorNTI, Invitrogen, Carlsbad, CA). Another alignment program is Sequencher(Gene Codes, Ann Arbor, Michigan), using default parameters.

As used herein, the term “nucleotide change” refers to, e.g., nucleotidesubstitution, deletion, and/or insertion, as is well understood in theart. For example, mutations contain alterations that produce silentsubstitutions, additions, or deletions, but do not alter the propertiesor activities of the encoded protein or how the proteins are made.

As used herein, the term “protein modification” refers to, e.g., aminoacid substitution, amino acid modification, deletion, and/or insertion,as is well understood in the art.

As used herein, the term “at least a portion” or “fragment” of a nucleicacid or polypeptide means a portion having the minimal sizecharacteristics of such sequences, or any larger fragment of the fulllength molecule, up to and including the full length molecule. Afragment of a polynucleotide of the disclosure may encode a biologicallyactive portion of a genetic regulatory element. A biologically activeportion of a genetic regulatory element can be prepared by isolating aportion of one of the polynucleotides of the disclosure that comprisesthe genetic regulatory element and assessing activity as describedherein. Similarly, a portion of a polypeptide may be 4 amino acids, 5amino acids, 6 amino acids, 7 amino acids, and so on, going up to thefull length polypeptide. The length of the portion to be used willdepend on the particular application. A portion of a nucleic acid usefulas a hybridization probe may be as short as 12 nucleotides; in someembodiments, it is 20 nucleotides. A portion of a polypeptide useful asan epitope may be as short as 4 amino acids. A portion of a polypeptidethat performs the function of the full-length polypeptide wouldgenerally be longer than 4 amino acids.

For PCR amplifications of the polynucleotides disclosed herein,oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from cDNA or genomic DNA extractedfrom any organism of interest. Methods for designing PCR primers and PCRcloning are generally known in the art and are disclosed in Sambrook etal. (2001) Molecular Cloning: A Laboratory Manual (3rd ed., Cold SpringHarbor Laboratory Press, Plainview, New York). See also Innis et al.,eds. (1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

The term “primer” as used herein refers to an oligonucleotide which iscapable of annealing to the amplification target allowing a DNApolymerase to attach, thereby serving as a point of initiation of DNAsynthesis when placed under conditions in which synthesis of primerextension product is induced, i.e., in the presence of nucleotides andan agent for polymerization such as DNA polymerase and at a suitabletemperature and pH. The (amplification) primer is preferably singlestranded for maximum efficiency in amplification. Preferably, the primeris an oligodeoxyribonucleotide. The primer must be sufficiently long toprime the synthesis of extension products in the presence of the agentfor polymerization. The exact lengths of the primers will depend on manyfactors, including temperature and composition (A/T vs. G/C content) ofprimer. A pair of bi-directional primers consists of one forward and onereverse primer as commonly used in the art of DNA amplification such asin PCR amplification.

The terms “stringency” or “stringent hybridization conditions” refer tohybridization conditions that affect the stability of hybrids, e.g.,temperature, salt concentration, pH, formamide concentration and thelike. These conditions are empirically optimized to maximize specificbinding and minimize non-specific binding of primer or probe to itstarget nucleic acid sequence. The terms as used include reference toconditions under which a probe or primer will hybridize to its targetsequence, to a detectably greater degree than other sequences (e.g. atleast 2-fold over background). Stringent conditions are sequencedependent and will be different in different circumstances. Longersequences hybridize specifically at higher temperatures. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (Tm) for the specific sequence at a defined ionicstrength and pH. The Tm is the temperature (under defined ionic strengthand pH) at which 50% of a complementary target sequence hybridizes to aperfectly matched probe or primer. Typically, stringent conditions willbe those in which the salt concentration is less than about 1.0 MNa+ion, typically about 0.01 to 1.0 M Na+ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes or primers (e.g. 10 to 50 nucleotides) and at least about60° C. for long probes or primers (e.g. greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. Exemplary low stringentconditions or “conditions of reduced stringency” include hybridizationwith a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. anda wash in 2×SSC at 40° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60° C. Hybridization procedures are well known in the art andare described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. Insome embodiments, stringent conditions are hybridization in 0.25 MNa2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecylsulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by awash in 5×SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C.to 65° C.

As used herein, the term “substantially identical” refers to twopolynucleotide sequences which vary in no more than 1, 2, 3, 4, 5, 6, or7 nucleotides. When used in the context of cTAGs, the term substantiallyidentical denotes two cTAGs that would be identical, except for amutation in the PAM or protospacer region of on one of the cTAGsdesigned to abrogate CRISPR cleavage in at least one CRISPR landingsite. When the term substantially identical is used in conjunction withthe term “partial” sequence or cTAG, the combination refers to thecomparison between two substantially identical cTAGs as described above,wherein one of the cTAGs has been digested by a CRISPR endonuclease.Thus the term would be used to indicate that the cTAG being describedwas identical to a second cTAG (in its undigested form), except for themutation in the PAM or protospacer region.

As used herein, the term “promoter” refers to a DNA sequence capable ofcontrolling the expression of a coding sequence or functional RNA. Thepromoter sequence may consist of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity, and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter.

As used herein, the term “heterologous” refers to a nucleic acidsequence which is not naturally found in the particular organism.

As used herein, the term “endogenous,” “endogenous gene,” refers to thenaturally occurring copy of a gene.

As used herein, the term “naturally occurring” refers to a gene orsequence derived from a naturally occurring source. In some embodimentsa naturally occurring gene refers to a gene of a wild type(non-transgene) gene, whether located in its endogenous setting withinthe source organism, or if placed in a “heterologous” setting, whenintroduced in a different organism. Thus, for the purposes of thisdisclosure, a “non-naturally occurring” sequence is a sequence that hasbeen synthesized, mutated, or otherwise modified to have a differentsequence from known natural sequences. In some embodiments, themodification may be at the protein level (e.g., amino acidsubstitutions). In other embodiments, the modification may be at the DNAlevel, without any effect on protein sequence (e.g., codonoptimization). In some embodiments, the non-naturally occurring sequencemay be a construct.

As used herein, the term “exogenous” is used interchangeably with theterm “heterologous,” and refers to a substance coming from some sourceother than its native source. For example, the terms “exogenousprotein,” or “exogenous gene” refer to a protein or gene from anon-native source or location, and that have been artificially suppliedto a biological system. Artificially mutated variants of endogenousgenes are considered “exogenous” for the purposes of this disclosure.

As used herein, the phrases “recombinant construct”, “expressionconstruct”, “chimeric construct”, “construct”, and “recombinant DNAconstruct” are used interchangeably herein. A recombinant constructcomprises an artificial combination of nucleic acid fragments, e.g.,regulatory and coding sequences that are not found together in nature.For example, a chimeric construct may comprise regulatory sequences andcoding sequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such constructmay be used by itself or may be used in conjunction with a vector. If avector is used then the choice of vector is dependent upon the methodthat will be used to transform host cells as is well known to thoseskilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the disclosure. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al., (1985) EMBOJ. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86),and thus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others. Vectors can be plasmids, viruses,bacteriophages, pro-viruses, phagemids, transposons, artificialchromosomes, and the like, that replicate autonomously or can integrateinto a chromosome of a host cell. A vector can also be a naked RNApolynucleotide, a naked DNA polynucleotide, a polynucleotide composed ofboth DNA and RNA within the same strand, a poly-lysine-conjugated DNA orRNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or thelike, that is not autonomously replicating. As used herein, the term“expression” refers to the production of a functional end-product e.g.,an mRNA or a protein (precursor or mature).

The term “operably linked” means in the context the sequentialarrangement of the promoter polynucleotide according to the disclosurewith a further oligo- or polynucleotide, resulting in transcription ofsaid further polynucleotide. In some embodiments, the promoter sequencesof the present disclosure are inserted just prior to a gene's 5′ UTR, oropen reading frame. In other embodiments, the operably linked promotersequences and gene sequences of the present disclosure are separated byone or more linker nucleotides. The term “operably linked” in thecontext of CRISPR protospacers and prospacer adjacent motifs (PAMs)refers to a proximately placed protospacer/PAM combination sequence thatis capable of being cleaved at high efficiency by a CRISPR endonucleasecomplex.

The term “CRISPR RNA” or “crRNA” refers to the RNA strand responsiblefor hybridizing with target DNA sequences, and recruiting CRISPRendonucleases. crRNAs may be naturally occurring, or may be synthesizedaccording to any known method of producing RNA.

The term “guide sequence” or “spacer” refers to the portion of a crRNAor guide RNA (gRNA) that is responsible for hybridizing with the targetDNA.

The term “protospacer” refers to the DNA sequence targeted by a crRNA orguide strand. In some embodiments, the protospacer sequence hybridizeswith the crRNA guide sequence of a CRISPR complex.

The term “seed region” refers to the ribonucleic sequence responsiblefor initial complexation between a DNA sequence CRISPR ribonucleoproteincomplex. Mismatches between the seed region and a target DNA sequencehave a stronger effect on target site recognition and cleavage than theremainder of the crRNA/sgRNA sequence. In some embodiments a singlemismatch in the seed region of a crRNA/gRNA can render a CRISPR complexinactive at that binding site. In some embodiments, the seed regions forCas9 endonucleases are located along the last ˜12 nt of the 3′ portionof the guide sequence, which correspond (hybridize) to the portion ofthe protospacer target sequence that is adjacent to the PAM. In someembodiments, the seed regions for Cpf1 endonucleases are located alongthe first ˜5 nt of the 5′ portion of the guide sequence, whichcorrespond (hybridize) to the portion of the protospacer target sequenceadjacent to the PAM.

The term “tracrRNA” refers to a small trans-encoded RNA. TracrRNA iscomplementary to and basepairs with crRNA to form a crRNA/tracrRNAhybrid, capable of recruiting CRISPR endonucleases to target sequences.

The term “Guide RNA” or “gRNA” as used herein refers to an RNA sequenceor combination of sequences capable of recruiting a CRISPR endonucleaseto a target sequence. Thus as used herein, a guide RNA can be a naturalor synthetic crRNA (e.g., for Cpf1), a natural or syntheticcrRNA/tracrRNA hybrid (e.g., for Cas9), or a single-guide RNA (sgRNA).

The term “CRISPR landing site” as used herein, refers to a DNA sequencecapable of being targeted by a CRISPR complex. Thus, in someembodiments, a CRISPR landing site comprises a proximately placedprotospacer/Protopacer Adjacent Motif combination sequence that iscapable of being cleaved a CRISPR endonuclease complex. The term“validated CRISPR landing site” refers to a CRISPR landing site forwhich there exists a guide RNA capable of inducing high efficiencycleaving of said sequence. Thus, the term validated should beinterpreted as meaning that the sequence has been previously shown to becleavable by a CRISPR complex. Each “validated CRISPR landing site” willby definition confirm the existence of a tested guide RNA associatedwith the validation.

The term “sticky end(s)” refers to double stranded polynucleotidemolecule end that comprises a sequence overhang. In some embodiments,the sticky end can be a dsDNA molecule end with a 5′ or 3′ sequenceoverhang. In some embodiments, the sticky ends of the present disclosureare capable of hybridizing with compatible sticky ends of the same orother molecules. Thus, in one embodiment, a sticky end on the 3′ of afirst DNA fragment may hybridize with a compatible sticky end on asecond DNA fragment. In some embodiments, these hybridized sticky endscan be sewn together by a ligase. In other embodiments, the sticky endsmight require extension of the overhangs to complete the dsDNA moleculeprior to ligation. The term “genetic scar(s)” refers to any undesirablesequence introduced into a nucleic acid sequence by DNA manipulationmethods. For example, in some embodiments, the present disclosureteaches genetic scars such as restriction enzyme binding sites, sequenceadapters or spacers to accommodate cloning, TA-sites, scars left overfrom NHEJ, etc. In some embodiments, the present disclosure teachesmethods of scarless cloning and gene editing.

As used herein the term “targeted” refers to the expectation that oneitem or molecule will interact with another item or molecule with adegree of specificity, so as to exclude non-targeted items or molecules.For example, a first polynucleotide that is targeted to a secondpolynucleotide, according to the present disclosure has been designed tohybridize with the second polynucleotide in a sequence specific manner(e.g., via Watson-Crick base pairing). In some embodiments, the selectedregion of hybridization is designed so as to render the hybridizationunique to the one, or more targeted regions. A second polynucleotide cancease to be a target of a first targeting polynucleotide, if itstargeting sequence (region of hybridization) is mutated, or is otherwiseremoved/separated from the second polynucleotide.

The disclosure refers to the taught and described universal modularCRISPR DNA constructs or designs as a “MegaModular” construct or design.

DNA Nucleases

In some embodiments, the present disclosure teaches methods andcompositions for gene editing/cloning utilizing DNA nucleases. CRISPRcomplexes, transcription activator-like effector nucleases (TALENs),zinc finger nucleases (ZFNs), and Fokl restriction enzymes are some ofthe sequence-specific nucleases that have been used as gene editingtools. These enzymes are able to target their nuclease activities todesired target loci through interactions with guide regions engineeredto recognize sequences of interest. In some embodiments, the presentdisclosure teaches CRISPR-based gene editing methods

The principles of in vivo CRISPR-based editing largely rely on naturalcellular DNA repair systems. Double-stranded dsDNA breaks introduced bynucleases are repaired by either non-homologous end-joining (NHEJ) orhomology-directed repair (HDR), or single strand annealing, (SSA), ormicrohomology end joining (MMEJ).

HDR relies on a template DNA containing sequences homologous to theregion surrounding the targeted site of DNA cleavage. Cellular repairproteins use the homology between the exogenously supplied or endogenousDNA sequences and the site surrounding a DNA break to repair the dsDNAbreak, replacing the break with the sequence on the template DNA.Failure to integrate the template DNA however, can result in NHEJ, MMEJ,or SSA. NHEJ, MMEJ and SSA are error-prone processes that are oftenaccompanied by insertion or deletion of nucleotides (indels) at thetarget site, resulting in genetic knockout (silencing) of the targetedregion of the genome due to frameshift mutations or insertions of apremature stop codon. Cpf1-mediated editing can also function viatraditional hybridization of overhangs created by the endonuclease,followed by ligation.

CRISPR endonucleases are also useful for in vitro DNA manipulations, asdiscussed in later sections of this disclosure.

CRISPR Systems

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) andCRISPR-associated (cas) endonucleases were originally discovered asadaptive immunity systems evolved by bacteria and archaea to protectagainst viral and plasmid invasion. Naturally occurring CRISPR/Cassystems in bacteria are composed of one or more Cas genes and one ormore CRISPR arrays consisting of short palindromic repeats of basesequences separated by genome-targeting sequences acquired frompreviously encountered viruses and plasmids (called spacers).(Wiedenheft, B., et. al. Nature. 2012; 482:331; Bhaya, D., et. al.,Annu. Rev. Genet. 2011; 45:231; and Terms, M. P. et. al., Curr. Opin.Microbiol. 2011; 14:321). Bacteria and archaea possessing one or moreCRISPR loci respond to viral or plasmid challenge by integrating shortfragments of foreign sequence (protospacers) into the host chromosome atthe proximal end of the CRISPR array. Transcription of CRISPR locigenerates a library of CRISPR-derived RNAs (crRNAs) containing sequencescomplementary to previously encountered invading nucleic acids(Haurwitz, R. E., et. al., Science. 2012:329;1355; Gesner, E. M., et.al., Nat. Struct. Mol. Biol. 2001:18;688; Jinek, M., et. al., Science.2012:337; 816-21). Target recognition by crRNAs occurs throughcomplementary base pairing with target DNA, which directs cleavage offoreign sequences by means of Cas proteins. (Jinek et. al. 2012 “AProgrammable dual-RNA-guided DNA endonuclease in adaptive bacterialimmunity.” Science. 2012:337; 816-821).

There are at least five main CRISPR system types (Type I, II, III, IVand V) and at least 16 distinct subtypes (Makarova, K. S., et al., NatRev Microbiol. 2015. Nat. Rev. Microbiol. 13, 722-736). CRISPR systemsare also classified based on their effector proteins. Class 1 systemspossess multi-subunit crRNA-effector complexes, whereas in class 2systems all functions of the effector complex are carried out by asingle protein (e.g., Cas9 or Cpf1). In some embodiments, the presentdisclosure teaches using type II and/or type V single-subunit effectorsystems. Thus, in some embodiments, the present disclosure teaches usingclass 2 CRISPR systems.

CRISPR/Cas9

In some embodiments, the present disclosure teaches methods of geneediting using a Type II CRISPR system. In some embodiments, the Type IICRISPR system uses the Cas9 enzyme. Type II systems rely on a i) singleendonuclease protein, ii) a transactiving crRNA (tracrRNA), and iii) acrRNA where a ˜20-nucleotide (nt) portion of the 5′ end of crRNA iscomplementary to a target nucleic acid. The region of a CRISPR crRNAstrand that is complementary to its target DNA protospacer is herebyreferred to as “guide sequence.”

In some embodiments, the tracrRNA and crRNA components of a Type IIsystem can be replaced by a single-guide RNA (sgRNA). The sgRNA caninclude, for example, a nucleotide sequence that comprises an at least12-20 nucleotide sequence complementary to the target DNA sequence(guide sequence) and can include a common scaffold RNA sequence at its3′ end. As used herein, “a common scaffold RNA” refers to any RNAsequence that mimics the tracrRNA sequence or any RNA sequences thatfunction as a tracrRNA.

Cas9 endonucleases produce blunt end DNA breaks and are recruited totarget DNA by a combination of a crRNA and a tracrRNA oligos, whichtether the endonuclease via complementary hybridization of the RNACRISPR complex. (see solid triangle arrows in FIG. 1A).

In some embodiments, DNA recognition by the crRNA/endonuclease complexrequires additional complementary base-pairing with a protospaceradjacent motif (PAM) (e.g., 5′-NGG-3′) located in a 3′ portion of thetarget DNA, downstream from the target protospacer. (Jinek, M., et. al.,Science. 2012:337;816-821). In some embodiments, the PAM motifrecognized by a Cas9 varies for different Cas9 proteins.

In some embodiments, one skilled in the art can appreciate that the Cas9disclosed herein can be any variant derived or isolated from any source.For example, in some embodiments, the Cas9 peptide of the presentdisclosure can include one or more of SEQ ID Nos selected from SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQID NO: 6. In other embodiments, the Cas9 peptide of the presentdisclosure can include one or more of the mutations described in theliterature, including but not limited to the functional mutationsdescribed in: Fonfara et al. Nucleic Acids Res. 2014February;42(4):2577-90; Nishimasu H. et al. Cell. 2014 Feb.27;156(5):935-49; Jinek M. et al. Science. 2012 337:816-21; and Jinek M.et al. Science. 2014 Mar. 14;343(6176); see also U.S. patent applicationSer. No. 13/842,859, filed Mar. 15, 2013, which is hereby incorporatedby reference; further, see U.S. Pat. Nos. 8,697,359; 8,771,945;8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308; 8,906,616;8,932,814; 8,945,839; 8,993,233; and 8,999,641, which are all herebyincorporated by reference. Thus, in some embodiments, the systems andmethods disclosed herein can be used with the wild type Cas9 proteinhaving double-stranded nuclease activity, Cas9 mutants that act assingle stranded nickases, or other mutants with modified nucleaseactivity.

CRISPR/Cpf1

In other embodiments, the present disclosure teaches methods of geneediting using a Type V CRISPR system. In some embodiments, the presentdisclosure teaches methods of using CRISPR from Prevotella andFrancisella 1 (Cpf1).

The Cpf1 CRISPR systems of the present disclosure comprise i) a singleendonuclease protein, and ii) a crRNA, wherein a portion of the 3′ endof crRNA contains the guide sequence complementary to a target nucleicacid. In this system, the Cpf1 nuclease is directly recruited to thetarget DNA by the crRNA (see solid triangle arrows in FIG. 1B). In someembodiments, guide sequences for Cpf1 must be at least 12nt, 13nt, 14nt,15nt, or 16nt in order to achieve detectable DNA cleavage, and a minimumof 14nt, 15nt, 16nt, 17nt, or 18nt to achieve efficient DNA cleavage.

The Cpf1 systems of the present disclosure differ from Cas9 in a varietyof ways. First, unlike Cas9, Cpf1 does not require a separate tracrRNAfor cleavage. In some embodiments, Cpf1crRNAs can be as short as about42-44 bases long—of which 23-25 nt is guide sequence and 19 nt is theconstitutive direct repeat sequence. In contrast, the combined Cas9tracrRNA and crRNA synthetic sequences can be about 100 bases long. Insome embodiments, the present disclosure will refer to a crRNA for Cpf1as a “guide RNA.”

Second, Cpf1 prefers a “TTN” PAM motif that is located 5′ upstream ofits target. This is in contrast to the “NGG” PAM motifs located on the3′ of the target DNA for Cas9 systems. In some embodiments, the uracilbase immediately preceding the guide sequence cannot be substituted(Zetsche, B. et al. 2015. “Cpf1 Is a Single RNA-Guided Endonuclease of aClass 2 CRISPR-Cas System” Cell 163, 759-771, which is herebyincorporated by reference in its entirety for all purposes).

Third, the cut sites for Cpf1 are staggered by about 3-5 bases, whichcreate “sticky ends” (Kim et al., 2016. “Genome-wide analysis revealsspecificities of Cpf1 endonucleases in human cells” published onlineJune 06, 2016). These sticky ends with -3-5 nt overhangs are thought tofacilitate NHEJ-mediated-ligation, and improve gene editing of DNAfragments with matching ends. The cut sites are in the 3′ end of thetarget DNA, distal to the 5′ end where the PAM is. The cut positionsusually follow the 18th base on the non-hybridized strand and thecorresponding 23rd base on the complementary strand hybridized to thecrRNA (FIG. 1B).

Fourth, in Cpf1 complexes, the “seed” region is located within the first5 nt of the guide sequence. Cpf1 crRNA seed regions are highly sensitiveto mutations, and even single base substitutions in this region candrastically reduce cleavage activity (see Zetsche B. et al. 2015 “Cpf1Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System” Cell163, 759-771). Critically, unlike the Cas9 CRISPR target, the cleavagesites and the seed region of Cpf1systems do not overlap. Additionalguidance on designing Cpf1 crRNA targeting oligos is available on(Zetsche B. et al. 2015. “Cpf1 Is a Single RNA-Guided Endonuclease of aClass 2 CRISPR-Cas System” Cell 163, 759-771).

Persons skilled in the art will appreciate that the Cpf1 disclosedherein can be any variant derived or isolated from any source. Forexample, in some embodiments, the Cpf1 peptide of the present disclosurecan include one or more of SEQ ID Nos selected from SEQ ID NO: 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, or any variants thereof.

Ligases

In some embodiments, the present disclosure teaches methods of cleavingtarget DNA via targeted Cpf1 complexes, and then ligating the resultingsticky ends with DNA inserts. In some embodiments, the presentdisclosure teaches methods of providing a Cpf1 complex to cleave thetarget DNA, and a ligase to “sew” the DNA back together. In otherembodiments, the present disclosure teaches modified Cpf1 complexes thatinclude a tethered ligase enzyme.

As used herein, the term “ligase” can comprise any number of enzymaticor non-enzymatic reagents. For example, ligase is an enzymatic ligationreagent or catalyst that, under appropriate conditions, formsphosphodiester bonds between the 3′-OH and the 5′-phosphate of adjacentnucleotides in DNA molecules, RNA molecules, or hybrids.

In some embodiments, the present disclosure teaches the use of enzymaticligases. Compatible temperature sensitive enzymatic ligases, include,but are not limited to, bacteriophage T4 ligase and E. coli ligase.Thermostable ligases include, but are not limited to, Afu ligase, Taqligase, Tfl ligase, Tth ligase, Tth HB8 ligase, Thermus species AK16Dligase and Pfu ligase (see for example Published P.C.T. ApplicationWO/2000/026381, Wu et al., Gene, 76(2):245-254, (1989), and Luo et al.,Nucleic Acids Research, 24(15): 3071-3078 (1996)). The skilled artisanwill appreciate that any number of thermostable ligases can be obtainedfrom thermophilic or hyperthermophilic organisms, for example, certainspecies of eubacteria and archaea; and that such ligases can be employedin the disclosed methods and kits. In some embodiments, reversiblyinactivated enzymes (see for example U.S. Pat. No. 5,773,258) can beemployed in some embodiments of the present teachings.

In other embodiments, the present disclosure teaches the use of chemicalligation agents. Chemical ligation agents include, without limitation,activating, condensing, and reducing agents, such as carbodiimide,cyanogen bromide (BrCN), N-cyanoimidazole, imidazole,1-methylimidazole/carbodiimide/cystamine, dithiothreitol (DTT) andultraviolet light. Autoligation, i.e., spontaneous ligation in theabsence of a ligating agent, is also within the scope of the teachingsherein. Detailed protocols for chemical ligation methods anddescriptions of appropriate reactive groups can be found in, among otherplaces, Xu et al., Nucleic Acid Res., 27:875-81 (1999); Gryaznov andLetsinger, Nucleic Acid Res. 21:1403-08 (1993); Gryaznov et al., NucleicAcid Res. 22:2366-69 (1994); Kanaya and Yanagawa, Biochemistry25:7423-30 (1986); Luebke and Dervan, Nucleic Acids Res. 20:3005-09(1992); Sievers and von Kiedrowski, Nature 369:221-24 (1994); Liu andTaylor, Nucleic Acids Res. 26:3300-04 (1999); Wang and Kool, NucleicAcids Res. 22:2326-33 (1994); Purmal et al., Nucleic Acids Res.20:3713-19 (1992); Ashley and Kushlan, Biochemistry 30:2927-33 (1991);Chu and Orgel, Nucleic Acids Res. 16:3671-91 (1988); Sokolova et al.,FEBS Letters 232:153-55 (1988); Naylor and Gilham, Biochemistry5:2722-28 (1966); and U.S. Pat. No. 5,476,930.

In some embodiments, the methods, kits and compositions of the presentdisclosure are also compatible with photoligation reactions.Photoligation using light of an appropriate wavelength as a ligationagent is also within the scope of the teachings. In some embodiments,photoligation comprises probes comprising nucleotide analogs, includingbut not limited to, 4-thiothymidine, 5-vinyluracil and its derivatives,or combinations thereof. In some embodiments, the ligation agentcomprises: (a) light in the UV-A range (about 320 nm to about 400 nm),the UV-B range (about 290 nm to about 320 nm), or combinations thereof,(b) light with a wavelength between about 300 nm and about 375 nm, (c)light with a wavelength of about 360 nm to about 370 nm; (d) light witha wavelength of about 364 nm to about 368 nm, or (e) light with awavelength of about 366 nm. In some embodiments, photoligation isreversible. Descriptions of photoligation can be found in, among otherplaces, Fujimoto et al., Nucl. Acid Symp. Ser. 42:39-40 (1999); Fujimotoet al., Nucl. Acid Res. Suppl. 1:185-86 (2001); Fujimoto et al., Nucl.Acid Suppl., 2:155-56 (2002); Liu and Taylor, Nucl. Acid Res. 26:3300-04(1998) and on the world wide web at: sbchem.kyoto-u.ac.jp/saito-lab.

Universal Modular CRISPR DNA Constructs and Uses Thereof

In some embodiments, the present invention describes a strategy for themodular assembly of DNA constructs. In some embodiments the DNA assemblymethods of the present disclosure are applicable to any construct,including plasmids, small linear DNA, and transformed chromosomal loci.

In aspects, the inventors refer to such a universal modular CRISPR DNAConstruct as a “MegaModular” design.

Shortcomings in Traditional DNA Editing and Assembly Techniques

Traditional multicomponent DNA cloning strategies are limited in theirability to effectively assemble and modify multi-component DNAconstructs with complex sequences. For example, restriction enzymecloning is limited by the availability of unique restriction enzymerecognition sites that are appropriately located at the cloningjunctures at each of the DNA inserts, and their destination sites withina final vector. Gateway cloning technologies are similarly limited bythe relatively small number of unique recombination sites available formulti-component assemblies.

Another downside to traditional DNA assembly techniques is that theirability to edit sequences is often restricted to the time ofconstruction. For example, the products of efficient assembly strategiessuch as Ligase Cycling Reactions (LCR are not easily modified once theinitial assembly is completed (Kok, S, et al., 2014 “Rapid and ReliableDNA assembly via Ligase Cycling Reaction” ACS Synth. Biol., 3 (2):97-106). Similar concerns arise with traditional restriction enzymecloning, whose common restriction recognition sites cease to function asunique cloning points once a polynucleotide containing the restrictionsites is inserted into the construct being assembled, or when saidconstruct is integrated into a chromosome full of said sites. Vectorsproduced through sequential restriction cloning thus provide very fewoptions for fixing or updating sequences once the cloning process iswell under way.

Even newer technologies, such as the traditional CRISPR DNA assemblytechniques continue to suffer from similar complexity, the ease ofiterating on a previous assembled construct/vector design, and speedlimitations (Wang, J W. et al., 2015 “CRISPR/Cas9 nuclease combined withGibson assembly for seamless cloning” BioTechniques, Vol 58, No.4:161-170). CRISPR cloning requires the design of a functional guide RNAtargeted next to a compatible protospacer adjacent motif (PAM).Availability of suitable PAM sequences within target sites results in asignificant design limitation to the number of possible DNA insertionlocations within a genome or construct.

Moreover, the design and testing of guide RNA sequences imposessignificant technical challenges for multi-component assemblies. Personshaving skill in the art will recognize for example, that not all gRNAsequences are functional, and that effective implementation of a CRISPRDNA assembly may sometimes require the design and validation of multiplegRNA sequence variants. These limitations are particularly cumbersome inmulti-component assemblies, where failure of a single gRNA sequence tosuccessfully produce a desired modification can trigger the need toredesign subsequent assembly components that no longer fall within theoriginal cloning plan. Applying techniques that require multiple customguide RNAs for every junction of a multicomponent assembly can thus alsobe very expensive, cumbersome, and impractical.

Modular CRISPR Tag Assembly Vectors and Methods of Using Such

In some embodiments, the present disclosure teaches methods for DNAassembly that overcome many of the limitations associated with theaforementioned traditional techniques described above. In someembodiments, the present disclosure also teaches modular CRISPR assemblyconstructs, compositions, and kits for use with the methods of thepresent invention.

In some embodiments, the present disclosure teaches DNA constructscomprising one or more CRISPR multi-clonal sites (cMCS). In someembodiments, the cMCS of the present disclosure represent only a portionof the DNA constructs described (i.e., only a portion of the constructis readily editable according to the methods of the present disclosure).In other embodiments, the cMCS of the present disclosure are located onkey positions within the entire construct, such that the entire DNAconstruct is readily editable. Thus, in some embodiments all thefunctional parts of the modular cTAG vectors (e.g., all origins,markers, cargo, elements required for assembly) are comprised withininsert DNA parts and can be readily exchanged via the gene editingmethods of the present disclosure.

In some embodiments, the cMCS of the present disclosure comprise one ormore cloning tags (cTAG), each comprising at least one validated CRISPRtargeting site. In some embodiments, the cMCS of the present disclosurefurther comprises DNA insert parts, each flanked by a pair of cTAGs,such that digestion of the cMCS with one or more CRISPR endonucleasetargeting one or more cTAGs, will release said flanked insert part,allowing for insertion of a compatible donor DNA part.

FIGS. 2 and 3 of this specification illustrate an embodiment of amodular CRISPR assembly plasmid construct, according to the methods ofthe present disclosure. The disclosed example plasmid contains a seriesof DNA insertions (Parts 1-8 in FIG. 2A), each flanked by a pair ofcTAGs (Tags A-H) in FIG. 2A. Digestion of cTAGs A and B of this examplewith the appropriate CRISPR/guide sequence complexes will release Part 2of the plasmid, allowing for insertion of a replacement part 2 insertwith the desired characteristics.

Persons having skill in the art will immediately recognize theadvantages of the presently described vector system, which allows forthe sequence-specific modular cloning/editing of vectors in vivo and invitro. The sections below will outline the various aspects of thedisclosed modular cloning vectors, as well as their various applicationsto molecular biology, gene therapy, and gene editing.

Modular CRISPR Vector Insert Parts

In some embodiments, the insert parts of the present disclosure aredonor DNA sequences for homologous recombination insertion following aCRISPR digestion. Thus, in some embodiments, insert part sequences ofthe present disclosure comprise an insert sequence of interest, flankedby sequences with sufficient homology to the ends of the digestedmodular CRISPR construct, so as to trigger homologous recombination,hybridization and insertion of the sequence.

In other embodiments, the insert parts of the present disclosure aredonor DNA sequences capable of hybridizing and ligation via sticky ends(e.g., following a Cpf1 digestion, restriction enzyme digestion, Gibsonassembly, or other hybridization-based assembly, including LCR). Thus,in some embodiments, insert part sequences of the present disclosurecomprise an insert sequence of interest, flanked by sequences withsufficient homology to the ends of the digested modular CRISPRconstruct, so as to allow for hybridization of sticky ends.

In yet other embodiments, the insert parts of the present disclosure aredonor DNA sequences for blunt end ligation.

In some embodiments, the modular CRISPR DNA constructs of the presentdisclosure are compatible with any insert part sequence. Thus, the partsof the present vectors can comprise, without limitation, selectablemarkers, origins of replication, promoters, terminator sequences; otherregulatory sequences, barcodes, recombination sites, or other sequencesof interest to the user. In some embodiments, the insert parts of thepresent disclosure can comprise homology sequences for triggeringhomologous recombination and insertion into one or more genetic loci. Insome embodiments, said homologous recombination insert parts willprecede and follow other insert parts that will be also be inserted intothe genome via the recombination event.

In some embodiments, the present disclosure teaches that each insertpart comprises a single sequence (e.g., only a promoter or only a geneof interest, see FIG. 2A, part 8). In other embodiments, the presentdisclosure teaches that one or more insert parts may contain multipleelements, such as promoter-gene of interest (GOI) combinations,multi-subunit chimeric protein fusions, or even entire constructs (seeFIG. 2A, part 5, comprising a promoter-GOI-terminator combination).

In some embodiments, the present disclosure teaches uncombinedindividual insert parts. That is, in some embodiments, the presentdisclosure teaches one or a plurality of unconnected insert parts (seeFIG. 2A, right side showing a list of uncombined insert parts). In someembodiments, the present disclosure teaches methods of assembling saidplurality of parts into one or more modular CRISPR constructs. In someaspects, the disclosure teaches kits for assembling a MegaModularconstruct.

In other embodiments, the present disclosure teaches partial- or fully-assembled modular CRISPR DNA constructs. For example, in someembodiments the present disclosure teaches modular CRISPR DNA constructscomprising 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more assembled insert parts,and any ranges therebetween. The disclosure also teaches kits comprisingsaid insert parts.

In some embodiments, said assembled or partially assembled modularCRISPR DNA constructs are linear. In some embodiments, said assembled orpartially assembled modular CRISPR DNA constructs are circular (e.g., aplasmid). In some embodiments, said assembled or partially assembledmodular CRISPR DNA constructs are integrated into genomic DNA.

In some embodiments, the constructs of the present disclosure willinitially contain only short spacer sequences as placeholders forfurther cloning (see “stuffer” sequence in FIG. 3C). In some embodimentsthe insert part placeholders are small, randomized sequences. In otherembodiments, the vectors of the present disclosure will initiallycomprise one or more pre-selected insert DNA parts. For example, in someembodiments, the modular CRISPR constructs will initially comprise atleast one selection marker, and/or at least one origin of replication.

Suitable selectable markers include, but are not limited to, genes thatconfer antibiotic resistance, genes that encode fluorescent proteins,tRNA genes, auxotrophic markers, toxic genes, phenotypic markers,antisense oligonucleotides, restriction endonucleases, restrictionendonuclease cleavage sites, enzyme cleavage sites, protein bindingsites, and sequences complementary to PCR primer sequences.

Suitable antibiotic resistance genes include, but are not limited to, achloramphenicol resistance gene, an ampicillin resistance gene, atetracycline resistance gene, a Zeocin resistance gene, a spectinomycinresistance gene and a kanamycin resistance gene.

In certain embodiments of the present invention, the counterselectablemarker is a toxic gene. Suitable toxic genes include, but are notlimited to, a ccdB gene, a gene encoding a tus protein which binds oneor more ter sites, a kicB gene, a sacB gene, an ASKI gene, a ΦX174 Egene and a DpnI gene. In some embodiments, the presence of a toxicselectable marker serves as an indicator that an insertion was notconducted, or was unsuccessful. Toxic selectable markers may also serveto decrease background of unmodified parent vectors of positive cells,by causing death to cells harboring unmodified vectors with the toxicgene still in place.

In additional embodiments of the methods of the present invention, themodular CRISPR constructs may comprise both one or more toxic genes andone or more antibiotic resistance genes.

In some embodiments, the modular CRISPR constructs will initiallycomprise at least one regulatory sequence. In some embodiments, thepresent disclosure teaches vectors comprising, without limitation,Matrix Attachment Regions, expression insulator sequences, expressionenhancer sequences, promoters, 5′ UTRs, 3′ UTRs, terminator sequences,stop codons, start codons, etc. In some embodiments, the modular CRISPRconstructs will initially comprise sequences for facilitatingchromosomal insertion of said construct (e.g., t-DNA borders, Cre/Lox,or homology ends to chromosomal sequences). In some embodiments, thesequences for chromosomal insertion are positioned so as to insert theentire modular CRISPR construct into the genome of an organism. In otherembodiments, the sequences for chromosomal insertion are positioned soas to insert only a portion of the modular CRISPR construct (see FIG.3D).

In some embodiments, the insert parts of the present disclosure can evencomprise additional cTAGs. The addition of cTAGs, through insert parts,can increase the complexity of available cloning schemes, and can alsoexpand the size of the construct by expanding the number of availableinsert parts that can be replaced.

In some embodiments, the insert parts of the present disclosure cancomprise a traditional cloning site. For example, in some embodiments,the present disclosure teaches insert parts comprising gatewayrecombination sites, restriction sites, Cre/Lox sites, or othertraditional cloning sites).

In some embodiments, the present disclosure teaches methods of producinginsert parts from traditional DNA constructs. That is, in someembodiments, the present disclosure teaches methods of adding cTAGs totraditional DNA constructs (e.g., to oligos, PCR fragments, plasmids, orother available DNA segment). In some embodiments, the presentdisclosure teaches methods of adding cTAGs to a single component, suchas a gene of interest (GOI), promoter. In other embodiments, the presentdisclosure teaches methods of adding cTAGS to multi-element constructs.

Persons having skill in the art will recognize methods for constructinginsert parts. For example, in some embodiments, the cTAGs may beincorporated into a DNA molecule via PCR amplification with primerscomprising said cTAGs. In other embodiments the cTAGs may beincorporated via traditional cloning techniques (e.g., restrictionenzymes, Gibson, or other assembly method). In yet other embodiments,the cTAGs can be incorporated via blunt-end ligation.

In some embodiments, the insert parts of the present disclosure can havea wide species compatibility spectrum (e.g., a marker may contain bothprokaryotic and eukaryotic expression sequences to make it effective inmultiple organisms). In other embodiments, the insert parts of thepresent disclosure are designed to have limited applicability toorganisms within a singlespecies/genus/family/order/class/phylum/kingdom or domain. In someembodiments for example, an origin of replication part may be capable ofmaintaining a plasmid in only a single species, or a group of species.In other embodiments, a fluorescent marker may be codon optimized tofunction across both prokaryotic and eukaryotic domains.

In some embodiments Cas9 endonucleases cleave 3-4 nucleotides upstreamfrom the PAM of a target sequence. cTAG digestion by a Cas9 complex canthus result in loss of cTAG functionality through the loss of the PAMsequence, or protospacer sequence of the target. In some embodiments,the present disclosure teaches methods of maintaining the functionalityof said cTAG sequences by designing donor insert sequences such thatthey reconstitute the cTAG sequence upon insertion (e.g., throughinsertion of the previously lost PAM of protospacer sequence). Similarprovisions are envisioned for sequences cleaved through Cpf1endonucleases.

FIG. 2B illustrates the presently disclosed concept of cTAG repair.Cleavage of insert part 2 with a Cas9 endonuclease also results in lossof a portion of cTAGs A and B. Subsequent insertions of any one ofinsert parts 2a-2d via homologous recombination results in a restorationof the full cTAG sequence.

Persons having skill in the art will recognize the nearly infiniteoptions for insert parts. The foregoing list of inserts was intended asillustrative, and should in no way be construed as limiting theapplicability of the presently disclosed methods, kits, and constructs.

Modular CRISPR Cloning Tags

In some embodiments the modular CRISPR constructs of the presentdisclosure comprise one or more cloning tags (cTAGs). In someembodiments, the modular CRISPR constructs of the present disclosurecomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more cTAGs.

In some embodiments, the present disclosure teaches that each cTAGcomprises at least one validated CRISPR protospacer/PAM combinationsequence (“CRISPR landing site”). That is, in some embodiments, cTAGscomprise at least one experimentally validated, high efficiency CRISPRlanding site. In some embodiments, the cTAGs of the present disclosuremay be validated by wet bench experimentation (e.g., in vitro cleavageof the cTAG sequence with a CRISPR complex targeting said CRISPR landingsite). In other embodiments, the cTAG validation may be assumed fromreports of cleavage in peer-reviewed journals.

In some embodiments, the cTAGs of the present disclosure comprise 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more CRISPR landing sites. In someembodiments, the CRISPR landing sites overlap with each other. In otherembodiments, the CRISPR landing sites occupy distinct non-overlappingregions within the cTAG. In some embodiments, the CRISPR landing sitescan be specific for either Cas9 or Cpf1 endonuclease cleavage. In someembodiments, the CRISPR landing sites can be specific to any othercurrent or yet to be discovered CRISPR endonuclease.

In other embodiments, the present disclosure teaches that multiplecloning sites in a single cTAG can be designed to function acrossdifferent organisms. Thus in some embodiments, cTAG Cpf1 landing sitesmay be preferred in organisms lacking or downregulating HR machinery. Inother embodiments, restriction sites of a cTAG may be preferred forinitial in vitro cloning, while Cas9 or Cpf1 landing sites may bepreferred for more complex editing occurring in vivo in selectedeukaryotic organisms.

In some embodiments, the present disclosure teaches that cTAGs maycomprise one or more non-CRISPR cloning sequences. For example, in someembodiments, the cTAGs of the present disclosure may comprise one ormore elements selected from the group consisting of a restriction enzymesite, a recombination site, a topoisomerase site, a splicing site, and aCre-Lox site.

In some embodiments, suitable restriction enzyme sites include, withoutlimitation, sites recognized by restriction enzymes selected from thegroup consisting of AaII, AarI, AasI, AatII, Acc65I, AccB7I, AccI,AccIII, AciI, AclI, AcuI, AdeI, AfeI, AflII, AflIII, AgeI, AhdI, AleI,AloI, AluI, Alw21I, Alw26I, Alw44I, AlwI, AlwNI, ApaI, ApaLI, ApeKI,ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeI, BalI, BamHI, BanI,BanII, BbsI, BbuI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BcnI,BcuI, BfaI, BfiI, BfmI BfrBI, BfuAI, BfuCI, BfuI, BglI, Bg1II, BlpI,Bme1390I, Bme1580I, BmgBI, BmrI, BmtI, BoxI, BpiI, BplI, BpmI, Bpu10I,Bpu1102I, BpuEI, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaMI, BsaWI, BsaXI,BseDI, BseGI, BseJI, BseLI, BseMI, BseMII, BseNI, BseRI, BseSI, BseXI,BseYI, BsgI, Bsh1236I, Bsh1285I, BshNI, BshTI, BsiEI, BsiHKAI, BsiWI,BsII, BsmAI, BsmBI, BsmFI, BsmI, BsoBI, Bsp19I, Bsp120I, Bsp1286I,Bsp1407I, Bsp143I, Bsp143II, Bsp68I, BspCNI, BspDI, BspEI, BspHI, BspLI,BspMI, BspPI, BspQI, BspTI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BsrSI,BssHII, BssKI, BssSI, Bst1107I, Bst98I, BstAPI, BstBI, BstEII, BstF5I,BstNI, BstOI, BstUI, BstXI, BstYI, BstZI, BstZ17I, Bsu15I, Bsu36I,BsuRI, BtgI, BtgZI, BtsCI, BtsI, BveI, Cac8I, CaiI, CfoI, Cfr10I,Cfr13I, Cfr42I, Cfr9I, CfrI, ClaI, CpoI, Csp45I, Csp6I, CspI, CspCI,CviaII, CviKI-1, CviQI, DdeI, DpnI, DpnII, DraI, DraIII, DrdI, EaeI,EagI, Eam1104I, Eam1105I, EarI, EciI, Ec1136II, EcIHKI, Eco105I,Eco130I, Eco147I, Eco24I, Eco31I, Eco32I, Eco47I, Eco47III, Eco52I,Eco57I, Eco57MI, Eco72I, Eco81I, Eco88I, Eco91I, EcolCRI, EcoNI,EcoO109I, EcoP15I, EcoRI, EcoRV, EbeI, Esp3I, FatI, FauI, Fnu4HI, FokI,FseI, FspI, FspAI, GsuI, HaeII, HaeIII, HgaI, HhaI, HinlI, Hin4I, Hin6I,HincII, HindIII, HinfI, HinP1I, HpaI, HpalI, HphI, Hpyl66II, Hpyl88I,Hpyl88III, Hpy8I, Hpy99I, HpyAV, HpyCH4III, HpyCH4IV, HpyCH4V, HpyF10VI,Hsp92I, Hsp921I, I-PpoI, KasI, Kpn2I, KpnI, KspAI, LweI, MbiI, MboI,MbolI, MfeI, MisI, MluI, MlyI, MmeI, MnII, Mph1103I, MscI, MseI, Ms1I,MspA1I, MspI, MssI, MunI, Mva1269I, MvaI, MwoI, NaeI, NarI, NciI, NcoI,NdeI, NdeII, NgoMIV, NheI, Nhel-HF, NlaIII, NlaIV, NmeAIII, NmuCI, NotI,NruI, NsbI, NsiI, NspI, OliI, PacI, PaeI, PaeR7I, PagI, PauI, PciI,PdiI, PdmI, Pf123II, PflFI, PflMI, PfoI, PhoI, PleI, PmeI, PmlI, PpiI,PpuMI, PshAI, PsiI, Psp1406I, Psp5II, PspGI, PspOMI, PspXI, PstI, PsuI,PsyI, PvuI, PvuII, PvuII-HF, RsaI, RsrII, SacI, SacII, SaII, SalI-HF,SapI, SatI, Sau3AI, Sau96I, SbfI, ScaI, ScaI-HF, SchI, ScrFI, SdaI,SduI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgfI, SgrAI, SinI, SmaI, SmiI,SmlI, SmuI, SnaBI, SpeI, SphI, SphI-HF, SspI, StuI, StyD4I, StyI, SwaI,TaaI, TaiI, TaqαI, TaqI, TasI, TatI, TauI, TfiI, TliI, Tru1I, Tru91,TseI, Tsp45I, Tsp509I, TspMI, TspRI, Tth111I, TurboNaeI, TurboNarI,Van91I, VspI, XagI, XapI, XbaI, XceI, XcmI, XhoI, XhoII, XmaI, XmaJI,XmiI, XmnI, and ZraI. Aspects also include homing endonucleases such as:I-Scel, I-Ceul, and PI-PspI. The corresponding cleavage sites for theseenzymes are known in the art.

In some embodiments the present disclosure teaches the use of rarerestriction enzymes, recognizing sites greater than or equal to eightnucleotides in length (≥8 restriction enzymes). In some embodiments, thepresent disclosure teaches use of a single rare restriction site in eachcTAG. In other embodiments, the cTAGs of the present disclosure maycomprise two or more restriction sites. Table 1 below provides a list ofcTAGs according to the present invention, each with their rarerestriction enzyme sites bolded.

TABLE 1Example cTAG sequences, CRISPR landing sites, and rare restrictionenzyme sites (bold sequence portions are restriction sites) TagAACTGGGTGGAATCCCTTCTGCAGCACCTGGATTACCCTGTTATCCCTAGT I-SceI SEQ ID NO: 65TagB TAATGAGTAGTCCTCATCTCCCTCAAGCAGGCGCCGGCGGTACTGCCATC MreI SEQ IDNO: 66 TagC CATATAATCTCCCTCAAGCAGGCCCCGCTGGCGCGCGCGAATGTTAGGAA MauBISEQ ID NO: 67 TagD GCCTATAATGTGAAGAGCTTCACTGAGTAGGGCCCGGGCTGTAAACGGTTSrfI SEQ ID NO: 68 TagEATTCGCTAGCAGATGTAGTGTTTCCACAGGGGCGATCGCTGATATGGGTC AsiSI SEQ ID NO: 69TagF ACTACCTAGCTGCATTTTCAGGAGGAAGCGATGGGCGGCCGCACACCTTC NotI SEQ IDNO: 70 TagG TGATAATGGGTGAGTGAGTGTGTGCGTGTGGGGCGCGCCAGATGGGAACA AscISEQ ID NO: 71 TagH ACTCCAGTCTTTCTAGAAGATGGCAAACAGCTATTATGGGTATTATGGGTPI- SEQ ID PspI NO: 72 TagITAGTGGACGGGGCCACTAGGGACAGGATTGGCCTGCAGGATTCCCGTCAA SbfI SEQ ID NO: 73TagJ TGAACTAAGGCGGCTGCACAACCAGTGGAG GCCTAAATGATC none SEQ ID NO: 74

In some embodiments, suitable recombination sites for use in the presentinvention include, but are not limited to: attB sites, attP sites, attLsites, attR sites, lox sites, psi sites, tnpI sites, dif sites, cersites, frt sites, and mutants, variants and derivatives thereof. Incertain embodiments of the present invention, the topoisomeraserecognition site, if present, is recognized and bound by a type Itopoisomerase, which may be a type IB topoisomerase. Suitable types oftype IB topoisomerase include, but are not limited to, eukaryoticnuclear type I topoisomerase and poxvirus topoisomerase. In someembodiments, suitable types of poxvirus topoisomerase include, but arenot limited to, poxvirus topoisomerase produced by or isolated from avirus such as vaccinia virus, Shope fibroma virus, ORF virus, fowlpoxvirus, molluscum contagiosum virus and Amsacta morreientomopoxvirus.

In some embodiments, cTAG arrangement of CRISPR and non-CRISPR cloningsites can be ordered according to user preference. In some embodiments,the present disclosure teaches that CRISPR binding sites should beordered so as to be the furthest away from insert parts. In oneillustrative embodiment, a cTAG could be arranged as follows from 5′-3′:(Part I)-[R1-A1-C-A2-R2]-(Part II), where R=restriction site,A=recombinase site, and C=CRISPR landing site. In some embodiments, Cmay include multiple overlapping, or sequential CRISPR and/orrestriction landing sites. In some embodiments, the arrangement ofcloning sites on a cTAG of the present disclosure will be symmetrical(i.e., provide for a symmetrical order of types of cloning sites).

In other embodiments, arrangement of cloning sites on a cTAG of thepresent disclosure may be non-symmetrical. For example, in anotherillustrative embodiment, a cTAG could be arranged as follows from 5′-3′:(Part I)-[R1-A1-C1-C2]-(Part II), where R=restriction site,A=recombinase site, and C1-2=CRISPR landing site(s). In yet otherembodiments, a cTAG could be arranged as follows from 5′-3′: i) (PartI)-[R1-C1-C2]-(Part II), ii) (Part I)-[R1-C1]-(Part II), iii) (PartI)-[C1-C2]-(Part II), or their reverse order, wherein R=restrictionsite, A=recombinase site, and C1-2=CRISPR landing site(s).

Persons having skill in the art will recognize the advantages andapplications of various cTAG arrangements. For example, in single-tagembodiments, the modular construct would allow for insertion with thedigestion of a single CRISPR endonuclease, but would not (without more,for example further digestion of additional cTAGs) allow for removal orreplacement of said insertion, due to the lack of a second flanking cTAGsite. In some embodiments, the present disclosure teaches that insertedparts may themselves contain additional cTAGs, to expand the number ofpossible insert part locations within the cMCS.

In other embodiments, the present disclosure teaches methods of removingone or more insert parts from the modular CRISPR constructs. In someembodiments, two or more of the cTAGs of a modular CRISPR constructcomprise restriction enzyme binding sites capable of creating compatibleends. In some embodiments, the restriction enzyme sites are identical.In other embodiments the restriction enzyme sites are distinct, but theresulting digestion of said sites produces compatible ends forhybridization and ligation. In some embodiments, the restriction sitesfor deletion of portions of a modular CRISPR construct are placed onother ends of two or more cTAGS, such that the resulting ligatedconstruct will still maintain the same ratio of insert parts to cTAGS.

In some embodiments, the present disclosure teaches that the restrictionenzyme sites used for deletions within the modular CRISPR constructs ofthe present disclosure can be any restriction enzyme that results incompatible ends. In other embodiments, the present disclosure teachesthat the restriction enzyme sites used for deletions within the modularCRISPR constructs of the present disclosure can be any rare 8≥baserestriction enzyme that result in compatible ends. In selectedembodiments, the present disclosure teaches that the restriction enzymesites used for deletions within the modular CRISPR constructs of thepresent disclosure can be I-SceI and PI-PspI.

In some embodiments, the present disclosure teaches modular CRISPRconstructs with two cTAGs flanking each insert part, so as to create acTAG pair. In some embodiments, the aforementioned cTAG pairs allow forthe selective cutting/replacement of insert parts. For example, asillustrated in FIG. 2B, digestion of the modular CRISPR plasmid withendonucleases targeting cTAGs A and B would result in the specificremoval of insert part 2.

As discussed above, selected embodiments of the present disclosureprovide for replacement insert parts which restore cTAG functionfollowing endonuclease cleavage. Thus, as illustrated in FIG. 2B,replacement insert parts 2a-2d comprise sequences that will restore cTAGA and B function upon insertion into the modular CRISPR plasmid.

In some embodiments, the present disclosure teaches that cTAGs can alsocontrol insert part directionality. Sequence homology between cTAG endsin insert parts and cleaved cTAGs in the modular CRISPR construct willdetermine insertion directionality for Cas9 cleaved sequences, eitherthrough homologous recombination or hybridization (e.g., in Gibsonapproaches). Insertion directionality in Cpf1 sequences may also becontrolled via Watson crick hybridization of Cpf1sticky ends on eithercTAG.

In some embodiments, the present disclosure also provides foralternative cTAG arrangements. For example, in some embodiments, themodular CRISPR constructs of the present disclosure may be designed suchas to provide functionality for the use of nested cTAGs.

In some embodiments, the present disclosure teaches component-basedCRISPR assemblies based on shared overlapping “tag” regions that enablemulticomponent assembly in vitro and in vivo. In some embodiments, thetags of the present disclosure comprise CRISPR landing sites tofacilitate future cloning or in vitro DNA assembly from DNA constructs.If DNA constructs are integrated into the genome of a host organism,preselected Cas9 or Cpf1 landing sites may facilitate facile geneticalterations. In a single suite of experiments, the assembly strategyenables construction of DNA plasmids that can be used in multipleorganisms, containing multiple numbers and types of DNA components.

In some embodiments, this assembly strategy can be used to assemble andquickly reassemble plasmids encoding any desired set of DNA components,including metabolic pathways. In other embodiments, designing cTAGs intointegrating plasmids can also be used to swap DNA components directly inand out of the genome of host organisms, circumventing the need to clonefuture plasmids.

cTAG Sequence Design Algorithm

In some embodiments, the present disclosure teaches algorithms designedto facilitate CRISPR landing sites within cTAGs. In some embodiments,the CRISPR landing sites are sequences identified from existingsequences. Thus, in some embodiments, the present disclosure teaches useof software programs is designed to identify candidate CRISPR targetsequences on both strands of an input DNA sequence based on desiredguide sequence length and a CRISPR motif sequence (PAM, protospaceradjacent motif) for a specified CRISPR enzyme. For example, target sitesfor Cpf1 from Francisella novicida U112, with PAM sequences TTN, may beidentified by searching for 5′-TTN- 3′ both on the input sequence and onthe reverse-complement of the input. The target sites for Cpf1 fromLachnospiraceae bacterium and Acidaminococcus sp., with PAM sequencesTTTN, may be identified by searching for 5′-TTTN-3′ both on the inputsequence and on the reverse complement of the input. Likewise, targetsites for Cas9 of S. thermophilus CRISPRI, with PAM sequence NNAGAAW,may be identified by searching for 5′-Nx-NNAGAAW-3′ both on the inputsequence and on the reverse-complement of the input. The PAM sequencefor Cas9 of S. pyogenes is 5′-NGG-3′.

Likewise, target sites for Cas9 of S. thermophilus CRISPR, with PAMsequence NGGNG, may be identified by searching for 5′-N, —NGGNG-3′ bothon the input sequence and on the reverse-complement of the input.

In other embodiments, the present disclosure teaches methods ofdesigning CRISPR landing sites from scratch. Persons having skill in theart will readily be able to design CRISPR landing sites in conjunctionwith the guide RNAs of the present disclosure, wherein the resultingprotospacer sequence is combined with the PAM motif appropriate to thedesired CRISPR endonuclease, as described above.

In some embodiments, the present disclosure teaches cTAGs comprising asequence selected from the group consisting of: SEQ ID NO. 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 78, 79, 80, 81, and combinations thereof.

Since multiple occurrences in the genome of the DNA target site may leadto nonspecific genome editing, after identifying all potential sites,the present disclosure teaches, in some embodiments, filtering outsequences based on the number of times they appear in the relevantreference genome or modular CRISPR construct. For those CRISPR enzymesfor which sequence specificity is determined by a ‘seed’ sequence (suchas the first 5 nt of the guide sequence for Cpf1-mediated cleavage) thefiltering step may also filter out different sequences with the sameseed.

In some embodiments algorithmic tools can also identify potential offtarget sites for a particular guide sequence. For example, in someembodiments Cas-Offinder can be used to identify potential off targetsites for Cpf1 (see Kim et al., 2016. “Genome-wide analysis revealsspecificities of Cpf1 endonucleases in human cells” published onlineJune 06, 2016). Any other publicly available CRISPRdesign/identification tool may also be used, including for example theZhang lab's crispr.mit.edu tool (see Hsu, et al. 2013 “DNA targetingspecificity of RNA_guided Cas9 nucleases” Nature Biotech 31, 827-832).

In some embodiments, the user may be allowed to choose the length of theseed sequence. The user may also be allowed to specify the number ofoccurrences of the seed:PAM sequence in a genome for purposes of passingthe filter. The default is to screen for unique sequences. Filtrationlevel is altered by changing both the length of the seed sequence andthe number of occurrences of the sequence in the genome. The programmay, in addition, or alternatively, provide the sequence of a guidesequence complementary to the reported target sequence(s) by providingthe reverse complement of the identified target sequence(s).

Modular CRISPR DNA Construct Cloning

In some embodiments, the present disclosure teaches methods forpreparing new recombinant nucleic acid molecules using the modularCRISPR DNA constructs of the present disclosure. In some embodiments,the present disclosure teaches methods of DNA part assembly.Descriptions of each method are provided below.

DNA Assembly Methods

In some embodiments, the present disclosure teaches methods for themodular assembly of DNA parts. In some embodiments, the DNA assemblymethods of the present disclosure are conducted in vitro. Thus, in someembodiments, the present disclosure teaches the steps of i) forming amixture comprising at least two insert part DNAs together with at leastone CRISPR complex, and ii) allowing said mixture to incubate inconditions for CRISPR digestion of the insert DNAs, iii) followed byhybridizing the compatible sticky ends from the digestion of each of thetwo insert part DNAs, and iv) ligating said hybridized ends to oneanother to create the new recombinant nucleic acid. Thus, in someembodiments, the insert part DNAs of the present disclosure are digestedtogether. In other embodiments, the present disclosure teaches methodsof digesting each insert part DNA individually, with the same ordifferent CRISPR complexes. In some embodiments, at least one insertpart is not digested by a CRISPR complex. In some embodiments, thepresent disclosure teaches that an exonuclease treatment is conductedprior to the hybridization of step iii) (for dual CRISPR digestions asdescribed in later sections).

In yet other embodiments, the present disclosure teaches Gibson-likejoining of insert parts, by exposing the insert part ends to an ssDNAexonuclease, and hybridizing the resulting sticky ends followed by anoptional fill with polymerase, and ligation. In some embodiments one ormore insert parts are exposed to a dsDNA exonuclease prior to the ssDNAexonuclease treatment. In some embodiments, the present disclosureteaches Gibson-like joining of insert parts or modular CRISPR vectorsthat have been digested by one or more CRISPR endonuclease (e.g., dualCRISPR digestions, as described in later sections).

The sections below provide a series of illustrative examplesdemonstrating the various ways in which the insert parts and modularCRISPR constructs of the present disclosure can be assembled and edited.The list of techniques described below provides an illustrative seriesof examples highlighting the utility of the sequences of the presentdisclosure, but is not intended to be limiting. Persons having skill inthe art will recognize other techniques that allow for the assembly andediting of insert parts according to the present disclosure.

In some embodiments, the present disclosure describes methods involvingCpf1 and/or Cas9 CRISPR endonucleases. Reference to these specificCRISPR endonucleases is illustrative, and is not intended to belimiting, unless specified in a claim. Persons having skill in the artwill immediately recognize the applicability of other existing—orheretofore undiscovered CRISPR endonucleases to the constructs andmethods of the present disclosure. References to Cpf1 may be interpretedas encompassing use of any presently known or undiscovered CRISPRendonuclease capable of catalyzing staggered DNA cleavage to producesticky DNA ends. References to Cas9 may similarly be interpreted asencompassing use of any presently known or undiscovered CRISPRendonuclease capable of catalyzing blunt end cleavage of dsDNA.

In Citro Cpf1

In some embodiments, the in vitro DNA assemblies of the presentdisclosure are conducted with Cpf1 CRISPR complexes as described below.First, two or more insert parts are incubated with a Cpf1 CRISPR complextargeting the cTAG that is common between the at least two insert parts.In some embodiments, the insert parts are incubated together in a singlemixture. In other embodiments, the insert parts are incubated indifferent mixtures.

Second, in some embodiments, the digested products are purified toremove active CRISPR nuclease. In some embodiments, the purificationinvolves separation of the active Cpf1 complex from the digested insertparts. In some embodiments, this can be accomplished through a DNApurification, such as a gel or column purification. In otherembodiments, the purification can be accomplished by Cpf1 inactivation,such as through heat or chemical inactivation.

Third, the digested insert parts are incubated in conditions appropriatefor hybridization of the compatible sticky ends created by the Cpf1complex. Hybridized ends are then ligated according to any knownligation methods, including those described in earlier portions of thisdisclosure.

In Vitro Cas9

In other embodiments, the in vitro DNA assemblies of the presentdisclosure are conducted with Cas9 CRISPR complexes as described below.First, two or more insert parts are incubated with a Cas9 CRISPR complextargeting the cTAG that is common between the at least two insert parts.In some embodiments, the insert parts are incubated together in a singlemixture. In other embodiments, the insert parts are incubated indifferent mixtures.

Second, in some embodiments, the digested products are purified toremove active CRISPR nuclease. In some embodiments, the purificationinvolves separation of the active Cas9 complex from the digested insertparts. In some embodiments, this can be accomplished through a DNApurification, such as a gel or column purification. In otherembodiments, the purification can be accomplished by Cas9 inactivation,such as through heat or chemical inactivation.

In some embodiments, the third step for Cas9 digested products is toincubate the insert parts in conditions appropriate for bluntend-ligation.

Dual CRISPR Assemblies

In other embodiments, the present disclosure also teachesGibson-assembly type methods for assembling the pieces ofCRISPR-digested insert parts with at least one shared cTAG sequence(e.g., assembly of compatible cTAGs digested at different CRISPR landingsites). Thus, in some embodiments, the present disclosure teaches dualCRISPR digestion assemblies as described below.

First, two or more insert parts are incubated with two CRISPR complexestargeting two different CRISPR landing sites flanking each part withinthe aforementioned cTAGs that are common between the at least two insertparts.

In some embodiments, the two different CRISPR landing sites are digestedtogether. In other embodiments, one insert part DNA is digested with oneCRISPR complex targeting one CRISPR landing site, and the other insertpart DNA is digested with a different CRISPR complex targeting thesecond CRISPR landing target site in separate vessels. In each case, theresult of these digestions will be that the shared cTAG in each of thetwo insert DNA cTAGs will comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 bp of sequence overlapwith each other.

For example, in an illustrative embodiment, the shared cTAG between twoinsert DNA parts would be arranged as follows from 5′-3′: (PartI)-[R1-C1-C2]-(Part II), where R=restriction site, C1=a first CRISPRlanding site and C2=a second CRISPR landing site. In this illustrativeembodiment, the first insert DNA part with a 3′ shared cTAG would bedigested with a CRISPR complex targeting C2 and the second insert DNApart with a 5′ shared cTAG would be digested with a CRISPR complextargeting C1. This would result in two DNA insert parts with overlappingsequence spanning C1-C2.

Second, in some embodiments, the digested products are purified toremove active CRISPR nuclease. In some embodiments, the purificationinvolves separation of the active CRISPR complex from the digestedinsert parts. In some embodiments, this can be accomplished through aDNA purification, such as a gel or column purification. In otherembodiments, the purification can be accomplished by CRISPRinactivation, such as through heat or chemical inactivation.

Third, in some embodiments, the CRISPR-digested insert parts areincubated with a ssDNA exonuclease to create overlapping sticky endsbetween the two insert DNA parts.

Fourth, the digested insert parts are incubated in conditionsappropriate for hybridization of the compatible sticky ends created bythe CRISPR complex/exonuclease digestions. Hybridized ends are thenligated according to any known ligation methods, including thosedescribed in earlier portions of this disclosure. In some embodiments,the hybridized parts are incubated with a polymerase to fill in anymissing sequence gaps prior to ligation.

Bridging Assemblies

In other embodiments, the present disclosure teaches Gibson-assembly ofCas9 digested parts, through the addition of a third DNA sequencecomprising a bridging sequence that overlaps with the digested cTAGsequences of the insert parts.

In this illustrative example, both insert parts are digested with thesame Cas9 CRISPR complex targeting the same CRISPR landing site. In thisembodiment, the resulting digested cTAGs would have no sequence overlap.Thus, in some embodiments, the third step is for Cas9 digested insertparts to be further digested with an ssDNA exonuclease to create either3′ or 5′ overhang. The exonuclease digested insert parts are thenincubated in conditions appropriate for hybridization of the compatiblesticky ends created by the combination of the CRISPR complex andexonuclease digestions with the bridging sequence. Hybridized ends arethen ligated according to any known ligation methods, including thosedescribed in earlier portions of this disclosure. In some embodimentsthe exonuclease digestion of the present disclosure is conducted beforethe second step.

In Vitro HDR

In other embodiments, the present disclosure teaches in vitro methods ofassembling the ends of insert part DNAs digested by a Cas9 or Cpf1endonuclease with an HDR complex, thereby triggering recombination ofsaid digested insert parts.

In Vivo Homologous Recombination

In some embodiments, the in vivo DNA assemblies of the presentdisclosure are conducted with Cpf1 or Cas9 CRISPR complexes as describedbelow. In one embodiment, two or more insert parts with at least oneshared cTAG are introduced into a host cell. In some embodiments, thepresence of DNA insert parts with homologous shared cTAG sequences willbe sufficient to trigger homologous recombination assembly (e.g., yeasthomologous recombination).

For example, in some embodiments, at least one shared cTAG sequencebetween the two insert DNA parts could be assembled to produce a linearconstruct. In this illustrative embodiment the two remaining outer cTAGscould also be designed to recombine with cTAGs of another vector withinthe cell (e.g., insertion into an existing plasmid, or a chromosome). Inother embodiments, the two parts could be further assembled into acircular construct through the recombination of a second shared cTAGbetween the two insert DNA parts. The assembled construct can be eitherused in the organism that was used for the assembly, or can, in someembodiments, be purified and transformed into a second organism (e.g.,assembly in yeast, and subsequent transformation into bacteria).

In other embodiments, one or more insert parts with a shared cTAG can bedigested prior to introduction into the host cell. Thus, in someembodiments, the present disclosure teaches CRISPR digestions to releaseinsert parts from larger vectors prior to in vivo assembly of thereleased parts. In some embodiments, the digestion is carried out withCas9. In other embodiments the digestion is carried out with Cpf1. Inother embodiments the digestion is carried out with restrictionendonucleases. In some embodiments the CRISPR digestions of insert partsare conducted in vitro. In some embodiments, the digested products arepurified to remove active CRISPR endonuclease prior to transformation ofthe insert parts into the assembly host cell.

In some embodiments, the purification step can be accomplished through aDNA purification, such as a gel or column purification. In otherembodiments, the purification can be accomplished by CRISPRinactivation, such as through heat or chemical inactivation.

In Vivo Ligation

In some embodiments, the present disclosure teaches methods ofprotecting insert parts from re-cleavage by the CRISPR endonuclease. Insome embodiments, the insert parts of the present disclosure may beprotected from endonuclease cleavage via chemical modification of theDNA sequence. For example, in some embodiments, the present disclosureteaches phosphorothioate oligonucleotides.

In some embodiments, the methods of the present disclosure areespecially useful for multi-part DNA assemblies.

FIG. 2A of the specification provides an illustrative example of amulti-part DNA assembly, according to the methods of the presentdisclosure. In this example, a series of eight DNA parts (parts 1-8),each with two cTAGs (tags A-H) are combined in vitro and are then ableto self-assemble (either via homologous recombination in vivo, or vialigation, as described above).

DNA Editing Methods

In some embodiments, the present disclosure teaches methods for theediting of modular CRISPR DNA constructs. In some embodiments, the DNAediting methods of the present disclosure apply the same principles ofthe DNA assembly methods described above, but do so for the purposes ofediting one or more pre-existing modular CRISPR DNA constructs.

In some embodiments, the DNA editing methods of the present disclosureare conducted in vitro. Thus, in some embodiments, the presentdisclosure teaches the steps of i) forming a mixture comprising amodular CRISPR DNA construct, and at least one insert DNA part, togetherwith at least one CRISPR complex, and ii) allowing said mixture toincubate in conditions for CRISPR digestion of the cTAGS of the insertDNA, and its corresponding modular CRISPR DNA construct cTAGs, followedby iii) hybridizing the compatible sticky ends (if Cpf1) produced by thedigestion of each of the aforementioned cTAGs, and iv) ligating saidhybridized ends (or blunt ends, if Cas9 is used) to one another tocreate the new recombinant nucleic acid. In some embodiments, anexonuclease treatment is conducted prior to the hybridization of stepiii) (for dual CRISPR digestions as described in later sections). Insome embodiments, the digestions of the present disclosure are conductedseparately for the insert part DNA and modular CRISPR DNA construct. Insome embodiments, only the modular CRISPR DNA construct is digested witha CRISPR complex.

In Vitro Cpf1

In some embodiments, the in vitro DNA editing methods of the presentdisclosure are conducted with Cpf1 CRISPR complexes as described below.First, a modular CRISPR DNA construct, and at least one insert DNA partare incubated with a Cpf1 CRISPR complex targeting the cTAGs of theinsert parts, and their corresponding tags within the modular CRISPR DNAconstruct. In some embodiments, the digestion of the modular CRISPR DNAand the insert part DNA is conducted in separate reactions.

Second, in some embodiments, the digested products are purified toremove active CRISPR nuclease. In some embodiments, the purificationinvolves separation of the active Cpf1 complex from the digestednucleotides. In some embodiments, this can be accomplished through a DNApurification, such as a gel or column purification. In otherembodiments, the purification can be accomplished by Cpf1 inactivation,such as through heat or chemical inactivation.

Third, the digested modular CRISPR DNA construct and insert parts areincubated in conditions appropriate for hybridization of the compatiblesticky ends created by the Cpf1complex. Hybridized ends are then ligatedaccording to any known ligation methods, including those described inearlier portions of this disclosure.

In Vitro Cas9

In other embodiments, the in vitro DNA editing methods of the presentdisclosure are conducted with Cas9 CRISPR complexes as described below.First, a modular CRISPR DNA construct, and at least one insert DNA partare incubated with a Cas9 CRISPR complex targeting the cTAGs of theinsert parts, and their corresponding tags within the modular CRISPR DNAconstruct. In some embodiments, the digestion of the modular CRISPR DNAand the insert part DNA are conducted in separate reactions.

Second, in some embodiments, the digested products are purified toremove active CRISPR nuclease. In some embodiments, the purificationinvolves separation of the active Cas9 complex from the digestednucleotides. In some embodiments, this can be accomplished through a DNApurification, such as a gel or column purification. In otherembodiments, the purification can be accomplished by Cas9 inactivation,such as through heat or chemical inactivation.

In some embodiments, the third step for Cas9 digested products is toincubate the insert parts in conditions appropriate for bluntend-ligation.

Gibson Editing

In other embodiments, the present disclosure also teachesGibson-assembly type methods for editing the sequences ofCRISPR-digested constructs and/or undigested insert parts containingintact overlapping cTAG sequence. Thus, in some embodiments, the thirdstep is for Cas9 digested modular CRISPR DNA construct and insertpart(s) to be further digested with an ssDNA exonuclease to createeither a 3′ or 5′ overhang. In some embodiments, the present disclosureteaches dsDNA exonuclease digestion to shorten the non-CRISPR digestedinsert parts prior to the ssDNA digestion.

The exonuclease digested DNA sections are then incubated underconditions appropriate for hybridization of the compatible sticky endscreated by the combination of the CRISPR complex and exonucleasedigestions. Hybridized ends are then ligated according to any knownligation methods, including those described in earlier portions of thisdisclosure. In some embodiments the hybridized DNA is incubated with apolymerase to fill in missing DNA sections prior to ligation. In someembodiments the exonuclease digestion of the present disclosure isconducted before the CRISPR inactivation step.

In some embodiments, the ligation of digested sequences can occur invitro.

In other embodiments, the present disclosure teaches in vitro methods ofassembling the ends of a modular CRISPR DNA construct digested by a Cas9or Cpf1 endonuclease, and at least one undigested insert with an HDRcomplex, thereby triggering recombination of said digested modularCRISPR DNA construct, and at least one insert DNA part.

In some embodiments of the DNA editing methods of the presentdisclosure, the DNA insert parts are comprised within a second modularCRISPR DNA construct. Thus, in some embodiments, the DNA editing methodsof the present disclosure comprise the transfer of a DNA insert partfrom one modular CRISPR DNA construct to another.

Expression, Purification, and Delivery

In some embodiments, the present disclosure teaches methods andcompositions of vectors, constructs, and nucleic acid sequences encodingCRISPR complexes. In some embodiments, the present disclosure teachesplasmids for transgenic or transient expression of the Cas9 orCpf1proteins. In some embodiments the present disclosure teaches aplasmid encoding chimeric Cas9 or Cpf1 proteins comprising in-framesequences for protein fusions of one or more of the other polypeptidesdescribed herein, including, but not limited to a ligase, a linker, andan NLS.

In some embodiments the plasmids and vectors of the present disclosurewill encode for the Cas9/Cpf1 protein(s) and also encode thecrRNA/tracrRNA/sgRNA, and/or donor insert sequences of the presentdisclosure. In other embodiments, the different components of theengineered complex can be encoded in one or more distinct plasmids.

In some embodiments, the plasmids of the present disclosure can be usedacross multiple species. In other embodiments, the plasmids of thepresent disclosure are tailored to the organism being transformed. Insome embodiments, the sequences of the present disclosure will becodon-optimized to express in the organism whose genes are being edited.Persons having skill in the art will recognize the importance of usingpromoters providing adequate expression for gene editing. In someembodiments, the plasmids for different species will require differentpromoters.

In some embodiments, the plasmids and vectors of the present disclosureare selectively expressed in the cells of interest. Thus in someembodiments, the present application teaches the use of ectopicpromoters, tissue-specific promoters, developmentally-regulatedpromoters, or inducible promoters. In some embodiments, the presentdisclosure also teaches the use of terminator sequences.

In some embodiments, the present disclosure also teaches methods ofexpressing and purifying Cpf1 and/or Cas9 endonuclease protein. In someembodiments, the present disclosure teaches that the proteins of thepresent disclosure may be produced by any of the commercially availableprotein production and purification kits or services. For example, insome embodiments, the present disclosure teaches methods of cloning Cas9and/or Cpf1 into a vector with a polyhistidine (His), glutathiones-transferase (GST), or other purification tag chimeric fusion. In someembodiments the present disclosure teaches a variety of prokaryotic andeukaryotic organisms, and cell-free protein production systems. Forexample, in some embodiments, the present disclosure teaches expressionof protein expression plasmids in E. coli BL21. In some embodiments, theprotein production system will be inducible, to reduce the effects ofprotein toxicity. For example, in some embodiments, the presentdisclosure teaches methods of using the IPTG or an arabinose inductionsystem.

In some embodiments, the present disclosure also teaches various proteinpurification schemes, including affinity tags (His-Nickel,GST-Glutathione, etc.). In some embodiments, the present disclosureteaches both native and denaturing conditions for protein purification.

In other embodiments, the present disclosure teaches production of Cas9and/or Cpf1 via one or more protein production services, including, butnot limited to GenScript®, ThermoFisher®, and NovoProtein®.

Transformation

In some embodiments, the present disclosure teaches the use oftransformation of the plasmids and vectors disclosed herein. Personshaving skill in the art will recognize that the plasmids of the presentdisclosure can be transformed into cells through any known system asdescribed in other portions of this specification. For example, in someembodiments, the present disclosure teaches transformation by particlebombardment, chemical transformation, agrobacterium transformation,nano-spike transformation, electroporation and virus transformation.

In some embodiments, the vectors of the present disclosure may beintroduced into the host cells using any of a variety of techniques,including transformation, transfection, transduction, viral infection,gene guns, or Ti-mediated gene transfer. Particular methods includecalcium phosphate transfection, DEAE-Dextran mediated transfection,lipofection, or electroporation (Davis, L., Dibner, M., Battey, I., 1986“Basic Methods in Molecular Biology”). Other methods of transformationinclude for example, lithium acetate transformation and electroporationSee, e.g., Gietz et al., Nucleic Acids Res. 27:69-74 (1992); Ito et al.,J. Bacterol. 153:163-168 (1983); and Becker and Guarente, Methods inEnzymology 194:182-187 (1991). In some embodiments, transformed hostcells are referred to as recombinant host strains.

In some embodiments, the present disclosure teaches high throughputtransformation of cells using the 96-well plate robotics platform andliquid handling machines of the present disclosure.

In some embodiments, the present disclosure teaches methods for gettingexogenous protein (Cpf1 /Cas9 and DNA ligase), RNA(crRNA/tracRNA/GuideRNA), and DNA (insert DNA part or modular CRISPRconstruct) into the cell. Various methods for achieving this have beendescribed previously including direct transfection of protein/RNA/DNA orDNA transformation followed by intracellular expression of RNA andprotein (Dicarlo, J. E. et al. “Genome engineering in Saccharomycescerevisiae using CRISPR-Cas systems.” Nucleic Acids Res (2013).doi:10.1093/nar/gkt135; Ren, Z. J., Baumann, R. G. & Black, L. W.“Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase:construction of a T4 phage hoc gene display vector.” Gene 195, 303-311(1997); Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. “Enhancedhomology-directed human genome engineering by controlled timing ofCRISPR/Cas9 delivery.” Elife 3, e04766 (2014)).

In some embodiments, the present disclosure teaches screeningtransformed cells with one or more selection markers as described above.In one such embodiment, cells transformed with a vector comprising akanamycin resistance marker (KanR) are plated on media containingeffective amounts of the kanamycin antibiotic. Colony forming unitsvisible on kanamycin-laced media are presumed to have incorporated thevector cassette into their genome. Insertion of the desired sequencescan be confirmed via PCR, restriction enzyme analysis, and/or sequencingof the relevant insertion site.

In other embodiments, a portion, or the entire complexes of the presentdisclosure can be delivered directly to cells. Thus, in someembodiments, the present disclosure teaches the expression andpurification of the polypeptides and nucleic acids of the presentdisclosure. Persons having skill in the art will recognize the many waysto purify protein and nucleic acids. In some embodiments, thepolypeptides can be expressed via inducible or constitutive proteinproduction systems such as the bacterial system, yeast system, plantcell system, or animal cell systems. In some embodiments, the presentdisclosure also teaches the purification of proteins and or polypeptidesvia affinity tags, or custom antibody purifications. In otherembodiments, the present disclosure also teaches methods of chemicalsynthesis for polynucleotides.

In some embodiments, persons having skill in the art will recognize thatviral vectors or plasmids for gene expression can be used to deliver thecomplexes disclosed herein. Virus-like particles (VLP) can be used toencapsulate ribonucleoprotein complexes, and purified ribonucleoproteincomplexes disclosed herein can be purified and delivered to cells viaelectroporation or injection.

Kits

In some embodiments, the disclosure provides kits containing any one ormore of the elements disclosed in the above methods and compositions. Insome embodiments, the kit comprises a modular CRISPR DNA construct andinstructions for using the kit and any necessary reagents or reactants.In some embodiments, the vector system comprises (a) a modular CRISPRDNA construct (b) a CRISPR complex, including a CRISPR endonucleaseprotein, and necessary target guide RNA(s) (or sequences encoding saiditems), and optionally (c) insert DNA parts, as describe supra in thisapplication.

Elements may be provided individually or in combinations, and may beprovided in any suitable container, such as a vial, a bottle, or a tube,or host cell, or plasmid. In some embodiments, the kit includesinstructions in one or more languages, for example in more than onelanguage.

In some embodiments, a kit comprises one or more reagents for use in aprocess utilizing one or more of the elements described herein (e.g.,purified Cpf1 endonuclease). Reagents may be provided in any suitablecontainer. For example, a kit may provide one or more reaction orstorage buffers. Reagents may be provided in a form that is usable in aparticular assay, or in a form that requires addition of one or moreother components before use (e.g. in concentrate or lyophilized form). Abuffer can be any buffer, including but not limited to a sodiumcarbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Trisbuffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In someembodiments, the buffer is alkaline. In some embodiments, the buffer hasa pH from about 7 to about 10. In some embodiments, the kit comprisesone or more oligonucleotides corresponding to a crRNA sequence forinsertion into a vector so as to operably link the crRNA sequence and aregulatory element.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the disclosure and are not meant to limit the presentdisclosure in any fashion. Changes therein and other uses which areencompassed within the spirit of the disclosure, as defined by the scopeof the claims, will occur to those skilled in the art.

Example 1 One-Pot In Vitro Modular CRISPR Cloning

This example describes the generation of plasmid 13001009086 (SEQ ID NO:82) by transfer of an insert from one plasmid to another in a one-potreaction. See, FIG. 4.

Both plasmids carry cloning tags flanking the region of interest (cTAG K[SEQ ID NO: 78] /cTAG L [SEQ ID NO: 79] and cTAG K′ [SEQ ID NO: 80]/cTAGL′ [SEQ ID NO: 81]). In order to drive the cloning reaction towards theedited plasmid, the Cpf1 spacers are in opposite orientations on therecipient and donor plasmids (K/K′ and L/L′ respectively). Thisinside-out/outside-in digest removes the Cpf1 spacer in the finalproduct, eliminating re-cutting of the desired product (see, curvedarrows in FIG. 4, depicting inside-out digestion in the '485 plasmid andoutside-in digestion in the '784 plasmid). The Cas9 spacers remain,enabling iterative editing at this site. Thus, the MegaModular constructallows for a rapid single-pot reaction scheme that enables iterativeediting.

Cpf1 protein was synthesized by Genscript and the crRNAs by Synthego.For the one-pot cleavage/ligation reaction, the Cpf1 protein complexedwith the crRNAs (crRNA 1 and crRNA 3), was added to the plasmids(13000789485—SEQ ID NO: 83 and 13000823784—SEQ ID NO: 84) and DNA ligasein buffer containing ATP. These components were cycled at temperaturesoptimized for cleavage and ligation.

The reaction was transformed into E. coli and positive clones weresequenced to confirm insertion of the new insert and loss of the Cpf1spacers.

For deletions, the Cpf1 sites within cloning tags used must generatecompatible overhangs to allow for plasmid closure. cTAG L′ was designedto contain two Cpf1 spacers, one for insertion where the overhang isincompatible with cTAG K′ and a second one for deletion where theoverhang is compatible with cTAG K′.

Example 2 In Vitro Modular CRISPR Cloning

This example was designed to demonstrate the flexibility of CRISPRcloning. As an initial step, several resistance plasmids encoding forKanamycin or Chloramphenicol resistance genes were created from sourcevectors pzHR039 (SEQ ID No: 100) and 13000223370 (SEQ ID No: 101),respectively. The Kanamycin resistance plasmids were each designed so asto include various Cpf1 landing sites flanking the GFP gene (whendigested, these plasmids produce “the kanamycin resistant plasmidbackbone”). The Chloramphenicol resistance plasmids were each designedso as to include various Cpf1 landing sites flanking the Chloramphenicolresistance gene (when digested, these plasmids produce “thechloramphenicol resistant insert”). Sequences, and vector maps for eachplasmid used in this Example are disclosed in Table 2.

Each Kanamycin and Chloramphenicol resistant plasmid was initiallylinearized with type-II restriction enzymes KpnI-HF and PvuI-HF,respectively (both commercially available from NEB). The location of theKpnI and PvuI restriction sites on each plasmid are noted in the vectormaps provided in FIGS. 7-14. After linearization, the resistanceplasmids were no longer capable of self-replication in a bacterial hostsystem.

Linearized resistance plasmids were then mixed with a pre-incubatedmixture of 15 ug (1.58 uM final concentration) of Cpf1 enzyme and 2 uLof 5 uM of each guide RNA described below (0.167 uM final concentration)in a 60 uL reaction to form active CRISPR complexes.

The Cpf1 enzyme used in this Example was commercially obtained from IDT.The Cpf1was sourced from Acidaminococcus sp. Cpf1 (AsCpf1). The enzymewas further modified to comprise 1 N-terminal nuclear localizationsequence (NLS) and 1 C-terminal NLSs, as well as 3 N-terminal FLAG tagsand a C-terminal 6-His tag.

The guide RNAs used in this example were custom ordered from IDT. Eachguide RNA was designed to target a different CRISPR landing site locatedwithin the linearized resistance plasmid. In this Example, the Cpf1landing sites of the backbone plasmid were excised, but restored uponligation of the insert. Table 2 provides the guide sequence portion ofeach guide RNA used. The CRISPR complexes in the mixture were thusdesigned to cleave out the GFP gene from each kanamycin resistantplasmid to generate kanamycin resistant plasmid backbones (see FIG. 5,second panel). The CRISPR complexes in the mixture were also designed tocleave out the chloramphenicol resistance gene from the chloramphenicolresistance plasmid to generate chloramphenicol resistant inserts (seeFIG. 5, second panel). The kanamycin resistant plasmid backbone and thechloramphenicol resistant insert of each reaction were similarlydesigned to generate compatible overhangs that would result inhybridization of the ends to produce a “dual resistant” kanamycin andchloramphenicol plasmid.

The linearized resistance plasmid mixtures comprising the Cpf1 and guideRNAs were allowed to incubate for 3 hours at 37 degrees Celsius in themanufacturer's recommended Cpf1buffer. Selected reactions were run onagarose gels and the resulting fragments were purified using standardDNA extraction kits (Zymo Research kit, used according to manufacturer'sinstructions). Purified (control) and unpurified (test).

DNA fragments comprising the kanamycin resistant plasmid backbone andthe chloramphenicol resistant insert, each comprising two compatibleCpf1 sticky ends were combined in a new reactions with or without a T4DNA ligase (commercially available form NEB) and transformed intoNEB10-B cells (commercially available from NEB). Transformed cells wereplated on media augmented with both Kanamycin and Chloramphenicoldesigned to prevent the growth of any cells that did not containfunctional resistance plasmids.

Individual colonies were sent for sequencing to confirm junctions ofCpf1 cloning. Recovered colonies were also validated via PCR usingprimers described in Table 2. FIG. 5 illustrates the generalexperimental design described above, except that the plasmids werelinearized prior to Cpf1 digestion, as described above.

TABLE 2 List of sequences used in this Example 2 Component DescriptionSEQ ID NO GFP Cpf1 cTAG M fwd 5′ SEQ ID No: 86CAGCACCTGGATTACCCTGTTATCCCTAGT TTTGGGTTAAAGATGGTTAAATGATTCGAAAATAATAAAGGGAAAATCA 3′ GFP Cpf1 cTAG N fwd 5′ SEQ ID No: 87CAGCACCTGGATTACCCTGTTATCCCTAGT TTTGGGATGTTAAGAGTCCCTATCTTCGAAAATAATAAAGGGAAAATCA 3′ GFP Cpf1 cTAG P fwd 5′ SEQ ID No: 88CAGCACCTGGATTACCCTGTTATCCCTAGT TTTGAGGAGTGTTCAGTCTCCGTGAACTCGAAAATAATAAAGGGAAAATCA 3′ GFP Cpf1 cTAG O rvs 5′ SEQ ID No: 89CGCTTCCTCCTGAAAATGCAGCTAGGTAGT TTTGACCGCCCCCCCCATACCCCAATCGACATGCCGAACTCAGAAGTGA 3′ GFP Cpf1 cTAG N rvs 5′ SEQ ID No: 90CGCTTCCTCCTGAAAATGCAGCTAGGTAGT TTTGGGATGTTAAGAGTCCCTATCTTCGACATGCCGAACTCAGAAGTGA 3′ CAT01 Cpf1 cTAG M fwd 5′ SEQ ID No: 91TTTGGGTTAAAGATGGTTAAATGATTCG ACATACACATAAAGTAGCTTGCG 3′CAT01 Cpf1 cTAG N fwd 5′ SEQ ID No: 92 TTTGGGATGTTAAGAGTCCCTATCTTCGACATACACATAAAGTAGCTTGCG 3′ CAT01 Cpf1 cTAG P fwd 5′ SEQ ID No: 93TTTGAGGAGTGTTCAGTCTCCGTGAACT CGACATACACATAAAGTAGCTTGCG 3′CAT01 Cpf1 cTAG N rvs 5′ SEQ ID No: 94 TTTGGGATGTTAAGAGTCCCTATCTTCGACTGGAAGGACAAGGGGGACC 3′ CAT01 Cpf1 cTAG O rvs 5′ SEQ ID No: 95TTTGACCGCCCCCCCCATACCCCAATCG ACTGGAAGGACAAGGGGGACC 3′ Cpf1 cTAG M5′ TTTGGGTTAAAGATGGTTAAATGAT 3′ SEQ ID No: 96 RNA targeting cTAG M 5′SEQ ID NO: 110 UAAUUUCUACUCUUGUAGAUGGUUAAAGAU GGUUAAAUGAU 3′ Cpf1 cTAG N5′ TTTGGGATGTTAAGAGTCCCTATCT 3′ SEQ ID No: 97 RNA targeting cTAG N 5′SEQ ID NO: 111 UAAUUUCUACUCUUGUAGAUGGAUGUUAAG AGUCCCUAUCU 3′ Cpf1 cTAG O5′ TTTGACCGCCCCCCCCATACCCCAA 3′ SEQ ID No: 98 RNA targeting cTAG O 5′SEQ ID NO: 112 UAAUUUCUACUCUUGUAGAUACCGCCCCCC CCAUACCCCAA 3′ Cpf1 cTAG P5′ TTTGAGGAGTGTTCAGTCTCCGTGAAC 3′ SEQ ID No: 99 RNA targeting cTAG P 5′UAAUUUCUACUCUUGUAGAUAGGAGUGUUC AGUCUCCGUGAAC 3′ SEQ ID NO: 113 pzHR039Source for Kanamycin resistance and GFP. SEQ ID NO: 100See listing for full sequence 13000223370Source for Chloramphenicol resistance and SEQ ID NO: 101GFP. See listing for full sequence pJDI427GFP Cpf1 cTAGs M and N KanR CENARS SEQ ID NO: 102TRP1 see listing for full sequence FIG. 7 pJDI429GFP Cpf1 cTAGs N and O KanR CENARS SEQ ID NO: 103TRP1 see listing for full sequence FIG. 8 pJDI430GFP Cpf1 cTAGs N and P KanR CENARS SEQ ID NO: 104TRP1 see listing for full sequence FIG. 9 pJDI431GFP Cpf1 cTAGs O and P KanR CENARS SEQ ID NO: 105TRP1 see listing for full sequence FIG. 10 pJDI432pJET AmpR CmR Cpf1 cTAGs M and N see SEQ ID NO: 106listing for full sequence FIG. 11 pJDI434pJET AmpR CmR blunt Cpf1 cTAGs N and O SEQ ID NO: 107see listing for full sequence FIG. 12 pJDI435pJET AmpR CmR blunt Cpf1 cTAGs N and P SEQ ID NO: 108see listing for full sequence FIG. 13 pJDI436pJET AmpR CmR blunt Cpf1 cTAGs O and P SEQ ID NO: 109see listing for full sequence FIG. 14 ***non-underlined portion of guideRNA for SEQ ID NOs: 110-113 is the chemically modified Alt-R RNA fromIDT. The homologous region of sequence to the respective cTAGs (i.e.M-P) is underlined.

The results of this experiment are shown in Table 3 and FIG. 6. Reactionnumbers for each transformation are shown along the top row, with guideRNAs used listed along the left-hand column of Table 3. The comparisonof identical Cpf1 reactions with and without ligase showed a 9.9-foldincrease in transformants in the presence of ligase enzyme, indicatingthat colony growth was due to formation of the double kanamycin andchloramphenicol resistant plasmid after Cpf1digestion. The no-ligasereactions are matched controls designed to establish that the reactionsare specific, and were not simply due to the presence of contaminatinglevels of undigested resistance plasmids.

Sixteen individual colonies were Sanger sequenced to verify both theupstream and downstream cloning junctions. In seven of seven upstreamsequenced junctions, and eight of nine downstream junctions, the Cpf1mediated clones from the reactions with T4 DNA ligase indicated faithfuldigestion and ligation.

Reactions 71 and 72 were transformed with Cpf1 digested plasmids thatwere not subjected to DNA gel purification steps. Cpf1 enzyme howeverwas heat inactivated according to supplier's instructions beforeaddition of T4 DNA ligase (reaction 72). Reactions 71 and 72 exhibitedthe same ligase-dependency.

TABLE 3 Resistant Transformant Colonies Comprising Cpf1-edited vectors55 56 59 60 67 68 71* 72* Guides M + N yes yes Guides N + O yes yesGuides P + O yes yes yes yes T4 Ligase No Yes No Yes No Yes No Yes # oftransformants 1 20 0 141 0 12 2 95 (Kan Resistant Colonies) *Plates 71and 72 were transformed with digested DNA that had not undergone DNA gelpurification after Cpf1 digestion.

The disclosure of PCT/US2017/042245 (WO 2018/013990 A1, claimingpriority to U.S. Provisional App. No. 62/362,909) is incorporated hereinin its entirety.

Example 3 Plasmid Assembly by Restriction Enzyme Digestion and LigationUsing the MegaModular Design

This example describes the genetic editing of a modular CRISPR vector,according to the methods of the present disclosure. FIG. 15 illustratesthe genetic editing of modular CRISPR plasmid 13000444591 described inthis example. The plasmid backbone was first prepared by removing a“stuffer” insert DNA part from a previously constructed plasmid. Thestuffer insert DNA part was removed by digesting the stuffer part'sflanking cloning tags (cTAGs) D (SEQ ID NO: 68) and E (SEQ ID NO: 69)with restriction enzymes ApaI and PvuI. The resulting fragments wereseparated via gel electrophoresis, and the desired 8.3 kb fragmentcorresponding to the plasmid backbone was excised from the gel andextracted using standard silica membrane columns.

To generate the new insert for the modular CRISPR vector, a desiredinsert DNA part flanked by cTAG D and cTAG E, was PCR amplified usinguniversal cTAG oligos tagD_FWD (SEQ ID NO: 75) and tagE_REV (SEQ ID NO:76). The resulting insert contained a GFP marker gene flanked by cTAG Dand cTAG E. The resulting PCR fragment was digested with the ApaI enzymethat cuts within cTAG D and the PvuI enzyme that cuts within cTAG Esequence. The digested insert DNA part was purified using standardsilica membrane columns.

The purified modular CRISPR vector backbone and insert DNA part werecombined into a single reaction with a ligase to generate a circularplasmid. The sequence for the resulting edited GFP-containing plasmid13000444591 is provided in (SEQ ID NO: 77).

Example 4 Plasmid Assembly by Yeast Homologous Recombination Using theMegaModular Design

Plasmid 13000283399 (SEQ ID NO: 85) was assembled by yeast homologousrecombination of PCR fragments flanked by MegaModular tags. The desiredconstructs for assembly were amplified by PCR in such a way that theywere flanked by specific MegaModular tags. These tags allowed fordirectional assembly of fragments in Saccharomyces cerevisiae as thetags themselves served as the overlapping homologous region forhomologous recombination.

Specifically, 5 fragments were amplified via PCR flanked by MegaModulartags as follows: tag A—Fragment 1—tag B; tag B—Fragment 2—tag C; tagC—Fragment 3—tag D; tag D—Fragment 4—tag E; and tag E—Fragment 5—tag F.These fragments, along with a linearized assembly vector containing ayeast origin of replication and a TRP auxotrophic selection marker aswell as tag A at one end and tag F at the other, were transformed intoS. cerevisiae. Circularized, assembled plasmids were selected by S.cerevisiae growth in media lacking tryptophan. These plasmids wererecovered and amplified in Escherichia coli, and correct conformationwas confirmed by sequencing.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes. However, mention of any reference,article, publication, patent, patent publication, and patent applicationcited herein is not, and should not, be taken as an acknowledgment orany form of suggestion that they constitute valid prior art or form partof the common general knowledge in any country in the world.

What is claimed is:
 1. A modular CRISPR DNA construct comprising aCRISPR multi-clonal site, said multi-clonal site comprising: a) aplurality of distinct cloning tags (cTAGs), said plurality of distinctcTAGs having been artificially introduced into the CRISPR DNA construct,wherein each cTAG comprises: i) a validated CRISPR landing sitecomprising a protospacer sequence operably linked to a protospaceradjacent motif (PAM); wherein the validated CRISPR landing site in atleast three of the plurality of distinct cTAGs is different from anyother CRISPR landing sites within the modular CRISPR DNA construct; andb) a plurality of distinct DNA inserts; i) wherein each DNA insert isflanked by a pair of cTAGs; and wherein the construct further comprisesat least 5 cTAGs.
 2. The modular CRISPR DNA construct of claim 1,wherein said modular CRISPR DNA construct is circular.
 3. The modularCRISPR DNA construct of claim 1, wherein said modular CRISPR DNAconstruct is linear.
 4. The modular CRISPR DNA construct of claim 1,wherein said modular CRISPR DNA construct is integrated into anorganism's genome.
 5. The modular CRISPR DNA construct of claim 1,wherein the validated CRISPR landing site is for a Cas9 endonuclease. 6.The modular CRISPR DNA construct of claim 1, wherein the validatedCRISPR landing site is for a Cpf1 endonuclease.
 7. The modular CRISPRDNA construct of claim 1, wherein at least one of said distinct cTAGscomprises a restriction endonuclease site at least 8 bases in length. 8.A reaction mixture comprising: A) a modular CRISPR DNA constructcomprising a plurality of distinct cloning tags (cTAGs), said pluralityof distinct cTAGS having been artificially introduced into the CRISPRDNA construct, wherein each cTAG comprises: i) a validated CRISPRlanding site(s), each comprising a protospacer sequence operably linkedto a protospacer adjacent motif (PAM); and B) a replacement DNA insert,wherein said replacement DNA insert is flanked by a first and secondinsert cTAG; i) wherein the first insert cTAG comprises the validatedCRISPR landing site of one of the distinct cTAGs of the CRISPR DNAconstruct, and the second insert cTAG comprises the validated CRISPRlanding site of another distinct cTAG of the CRISPR DNA construct; andC) a first and second CRISPR complex targeting the first and secondinsert cTAGs, respectively, each CRISPR complex comprising: i) a CRISPRendonuclease, and ii) a guide RNA capable of recruiting said CRISPRendonuclease to one of said targeted insert cTAGs.
 9. The reactionmixture of claim 8, wherein said modular CRISPR DNA construct iscircular.
 10. The reaction mixture of claim 8, wherein said modularCRISPR DNA construct is linear.
 11. The reaction mixture of claim 8,wherein said modular CRISPR DNA construct is integrated into anorganism's genome.
 12. The reaction mixture of claim 8, wherein theCRISPR endonuclease is Cas9.
 13. The reaction mixture of claim 8,wherein the CRISPR endonuclease is Cpf1.
 14. The reaction of claim 8,wherein at least one of said distinct cTAGs comprises a restrictionendonuclease site at least 8 bases in length.
 15. The reaction of claim8, wherein the guide RNA comprises a tracrRNA and a crRNA.
 16. Areaction mixture comprising: A) a plurality of different DNA inserts,wherein each DNA insert is flanked by a pair of artificially introducedcloning tags (cTAGs), each cTAG comprising: i) a validated CRISPRlanding site(s), each comprising a protospacer sequence operably linkedto a protospacer adjacent motif (PAM); and ii) one or more CRISPRcomplexes targeting at least one of the cTAGs present in at least two ofthe plurality of different DNA inserts, each CRISPR complex comprising:a) a CRISPR endonuclease, and b) a guide RNA capable of recruiting saidCRISPR endonuclease to one of said targeted cTAGs.
 17. The reactionmixture of claim 16, wherein the CRISPR endonuclease is Cas9.
 18. Thereaction mixture of claim 16, wherein the CRISPR endonuclease is Cpf1 .19. The reaction mixture of claim 16, wherein at least one of the cTAGscomprises a restriction endonuclease site at least 8 bases in length.20. The reaction mixture of claim 16, wherein the guide RNA comprises atracrRNA and a crRNA.